Mixed-species Experiments Research Articles

Harnessing HetHydrogel: A Universal Platform to Dropletize Single-Cell Multiomics.

A universal platform is developed for dropletizing single cell plate-based multiomic assays, consisting of three main pillars: a miniaturized open Heterogeneous Hydrogel reactor (abbreviated HetHydrogel) for multi-step biochemistry, its tunable permeability that allows Tn5 tagmentation, and single cell droplet barcoding. Through optimizing the HetHydrogel manufacturing procedure, the chemical composition, and cell permeation conditions, simultaneous high-throughput mitochondrial DNA genotyping and chromatin profiling at the single-cell level are demonstrated using a mixed-species experiment. This platform offers a powerful way to investigate the genotype-phenotype relationships of various mtDNA mutations in biological processes. The HetHydrogel platform is believed to have the potential to democratize droplet technologies, upgrading a whole range of plate-based single cell assays to high throughput format.

Opto-combinatorial indexing enables high-content transcriptomics by linking cell images and transcriptomes.

We introduce a simple integrated analysis method that links cellular phenotypic behaviour with single-cell RNA sequencing (scRNA-seq) by utilizing a combination of optical indices from cells and hydrogel beads. With our method, the combinations, referred to as joint colour codes, enable the link via matching the optical combinations measured by conventional epi-fluorescence microscopy with the concatenated DNA molecular barcodes created by cell-hydrogel bead pairs and sequenced by next-generation sequencing. We validated our approach by demonstrating an accurate link between the cell image and scRNA-seq with mixed species experiments, longitudinal cell tagging by electroporation and lipofection, and gene expression analysis. Furthermore, we extended our approach to multiplexed chemical transcriptomics, which enabled us to identify distinct phenotypic behaviours in HeLa cells treated with various concentrations of paclitaxel, and determine the corresponding gene regulation associated with the formation of a multipolar spindle.

A CRISPR-based strategy for targeted sequencing in biodiversity science.

Many applications in molecular ecology require the ability to match specific DNA sequences from single- or mixed-species samples with a diagnostic reference library. Widely used methods for DNA barcoding and metabarcoding employ PCR and amplicon sequencing to identify taxa based on target sequences, but the target-specific enrichment capabilities of CRISPR-Cas systems may offer advantages in some applications. We identified 54,837 CRISPR-Cas guide RNAs that may be useful for enriching chloroplast DNA across phylogenetically diverse plant species. We tested a subset of 17 guide RNAs invitro to enrich plant DNA strands ranging in size from diagnostic DNA barcodes of 1,428 bp to entire chloroplast genomes of 121,284 bp. We used an Oxford Nanopore sequencer to evaluate sequencing success based on both single- and mixed-species samples, which yielded mean chloroplast sequence lengths of 2,530-11,367 bp, depending on the experiment. In comparison to mixed-species experiments, single-species experiments yielded more on-target sequence reads and greater mean pairwise identity between contigs and the plant species' reference genomes. But nevertheless, these mixed-species experiments yielded sufficient data to provide ≥48-fold increase in sequence length and better estimates of relative abundance for a commercially prepared mixture of plant species compared to DNA metabarcoding based on the chloroplast trnL-P6 marker. Prior work developed CRISPR-based enrichment protocols for long-read sequencing and our experiments pioneered its use for plant DNA barcoding and chloroplast assemblies that may have advantages over workflows that require PCR and short-read sequencing. Future work would benefit from continuing to develop invitro and in silico methods for CRISPR-based analyses of mixed-species samples, especially when the appropriate reference genomes for contig assembly cannot be known a priori.

Optimizing height measurement for the long-term forest experiments in Sweden

Information on tree height is useful for volume estimation and site productivity assessment and as such, remains one of the most important variables often measured in forest inventories. Measuring a sufficient number of sample trees requires considerable sampling effort and cost. In this study, we developed height functions for optimizing tree height measurement in the Swedish long-term forest experiments (LTFEs). Two large datasets from the LTFE databases: fitting data (from thinning, fertilisation and mixed species experiments) and validation data (tree species and spacing experiments) collected over several decades were used. The fitting and validation data comprise 133,788 and 68,440 observations, respectively, each covering a large range of growth and environmental conditions across Sweden. A multilevel nonlinear mixed-effects modelling approach was used to build the generalised height functions for Scots pine, Norway spruce, birch (Silver and Downy birch united), other conifers and other broadleaves, considering variations in heights and other stand characteristics at sample plot-level and revision-level. The response calibration of the functions was first carried out with all measured heights of the validation data, and second, using heights of one to six sample trees obtained from different tree selection strategies (diameter extremes, largest diameters, and smallest diameters). The mixed-effects height functions explained most of the height variations in the fitting dataset (pseudo R2: 0.938 – 0.970; RMSE: 0.957 – 1.363 m) without any residual trends. The validation showed that the functions accounted for 95 – 98 % of the height variation in the validation dataset, with RMSE ranging between 0.770 and 1.040 m, confirming the functions’ high accuracy. We recommend the measurement of four sample tree heights based on diameter extremes as the ideal threshold for response calibration. These functions and the suggested sampling technique would reduce sampling effort and inventory cost of height measurements for subsequent inventories of the LTFEs.

Onepot-Seq: capturing single-cell transcriptomes simultaneously in a continuous medium via transient localization of mRNA.

The development of single-cell RNA-seq has broadened the spectrum for biological research by providing a high-resolution analysis of cellular heterogeneity. However, the requirement for sophisticated devices for the compartmentalization of cells has limited its widespread applicability. Here, we develop Onepot-Seq, a device-free method, that harnesses the transient localization of mRNA after lysis to capture single-cell transcriptomes simultaneously in a continuous fluid medium. In mixed-species experiments, we obtained high-quality single-cell profiles. Further, cell type-specific poly(A)-conjugated antibodies allow Onepot-Seq to effectively capture target cells in complex populations. Chemical perturbations to cells can be profiled by Onepot-Seq at single-cell resolution. Onepot-Seq should allow routine transcriptional profiling at single-cell resolution, accelerating clinical and scientific discoveries in many fields of science.

Competing Sorption of Se(IV) and Se(VI) on Schwertmannite

Schwertmannite (SHM) is a naturally occurring mineral that has been shown to effectively scavenge oxyanions from contaminated water. In this study, Fourier-transform infrared spectroscopy and X-ray absorption spectroscopy techniques in combination with wet-chemical techniques were used to study the competitive sorption of Se(IV) and Se(VI) at pH 3. The experiments were conducted with three types of schwertmannite obtained from oxidative synthesis, biogenic synthesis and high-pressure compaction at different initial Se concentrations and mixing ratios for 48 h and 56 days, respectively. A threshold value for the uptake mechanisms was identified, which reflects the amount of easily exchangeable sulphate (~0.5 mmol/g). At adsorbate concentrations below this threshold, an inner-sphere corner-sharing bidentate binuclear complex forms upon exchange with sulphate. At higher concentrations, both oxyanions become bound to SHM through co-occurrence of mainly inner-sphere and partly outer-sphere corner-sharing bidentate binuclear complexes with Fe(III) containing surface sites. Single species experiments clearly indicate a higher affinity of SHM for Se(IV). However, in mixed species experiments, competitive sorption occurs with equal or even preferential uptake of Se(VI) at concentrations much lower than the threshold value, presumably due to geometrical similarity between selenate and sulphate, and increasing preference for Se(IV) at high Se concentrations.

On the use of observational data in studying biodiversity-productivity relationships in forests

The biodiversity-productivity relationship is one of the focus areas in ecological research that is studied primarily through mixed species experiments. Recent efforts in forests, however, increasi...

Mixed stands of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst] can be more productive than monocultures. Evidence from over 100 years of observation of long-term experiments

Aim of study: The objective of this study was to analyse the effect of species mixing of Scots pine and Norway spruce on the productivity at the stand and species level. We also analysed to what extent the mixing effects is modified by drought stress.Area of study: The study was conducted inN-E Poland and based on three experiments located in Maskulińskie, Strzałowo and Kwidzyn Forest Districts.Material and methods: We evaluated long-term mixed-species experiments in Scots pine and Norway spruce which are under continuous survey since more than 100 years. Stand productivity was analysed based on the periodic annual increment and total yield of stem volume. Growth and yield were compared between mixed and neighbouring pure stands. As a substitute for the missing Norway spruce monocultures, we used appropriate yield table data. In order to characterize the effect of water supply on the mixing effects, we correlated the Martonne index of aridity with the ratio of Scots pine growth in mixed versus pure stands.Main results: We found that the mixed stands exceed the weighted mean of the pure stands’ volume productivity on average by 41%. At the species level Scots pine benefits from the mixture by 34% and Norway spruce by 83%. Growth periods with harsh climate conditions reinforce overyielding, while periods with mild conditions reduce the benefit of mixing. The overyielding of mixed stands, especially when growing under unfavourable conditions, is explained by niche complementarity of both species and discussed in view of the stress-gradient-hypothesis.Research highlights: The revealed overyielding of mixed compared with neighbouring pure stands, particularly under harsh weather conditions, substantiates the preferences of Scots pine-Norway spruce mixtures regarding climate change. Keywords: drought resilience; mixed stand; pure stand; facilitation; competition; overyielding; underyielding.

Influence of substrate preference and complexity on co-existence of two non-native gammarideans (Crustacea: Amphipoda)

Substrate choice, swimming activity and risk to predation by burbot (Lota lota) of the well established Gammarus roeselii and the invader Dikerogammarus villosus were studied in mixed and single-species aquarium experiments. We used stones, gravel and aquatic weeds (Elodea, Chara) as substrates. We hypothesized that both species have different substrate preferences and that substrate affects the predation risk. We also assumed that presence of D. villosus influences substrate preference and predation risk of G. roeselii since the invader is known to affect the behavior of other gammarids. Adults of D. villosus in single species experiments and juveniles in mixed and single species experiments were evenly distributed over the different substrates but adults in mixed species experiments were more likely to prefer stone substrate. In contrast, adults and juveniles of G. roeselii clearly preferred aquatic weeds independent of the presence/absence of the invader. Both species preferred substrates with fissured surface over substrates with smooth surface. Gammarus roeselii was observed swimming more often than D. villosus in the open water but its swimming activity was lower when its preferred substrate was present compared with its swimming activity if non-preferred substrates were present. Predation rate of burbot on D. villosus was comparatively low and independent of the substrate. Burbot consumed many more G. roeselii than D. villosus, both in mixed and single species experiments. But when the preferred substrate of G. roeselii (weeds) was used in the experiments, predation rate of burbot on G. roeselii was somewhat lower than that when non-preferred substrates were present. The results of the experiments support our hypothesis that the gammarids studied here have different substrate preferences and that presence of the preferred substrate can affect predation risk. However, there is no evidence that presence of D. villosus affected substrate choice or predation risk in G. roeselii. We consider that differences in use of spatial niches permit co-existence of G. roeselii and D. villosus in the wild when substrates are diverse. The fact that G. roeselii than D. villosus is more often observed swimming in the open water may explain its higher risk of being captured by fish.

Modelling predicts positive and negative interactions between three Australian tropical tree species in monoculture and binary mixture

Computer modelling promises to be an important tool for analysing and predicting interactions between trees within mixed species forest plantations. This study explored the use of an individual-based mechanistic model as a predictive tool for designing mixed species plantations of Australian tropical trees. The ‘spatially explicit individually based-forest simulator’ (SeXI-FS) modelling system was used to describe the spatial interaction of individual tree crowns within a binary mixed-species experiment. The three-dimensional model was developed and verified with field data from three forest tree species grown in tropical Australia. The model predicted the interactions within monocultures and binary mixtures of Flindersia brayleyana, Eucalyptus pellita and Elaeocarpus grandis, accounting for an average of 42% of the growth variation exhibited by species in different treatments. The model requires only structural dimensions and shade tolerance as species parameters. By modelling interactions in existing tree mixtures, the model predicted both increases and reductions in the growth of mixtures (up to ±50% of stem volume at 7 years) compared to monocultures. This modelling approach may be useful for designing mixed tree plantations.

The Effects of Zebra Mussels (Dreissena polymorpha) on the Foraging Success of Eurasian Perch (Perca fluviatilis) and Ruffe (Gymnocephalus cernuus)

AbstractComplex habitat structures can influence the foraging success of fish. Competition for food between fish species can therefore depend on the competitors' abilities to cope with structural complexity. In laboratory experiments, we comparatively assessed effects of zebra mussels (Dreissena polymorpha Pall.) on the foraging success of Eurasian perch (Perca fluviatilis L.) and ruffe (Gymnocephalus cernuus (L.)). In single‐species and mixed‐species experiments, the fish were fed caddisfly larvae (Tinodes waeneri (L.)) over complex (mussel‐covered stones) and less‐complex (bare stones) substrates. With intraspecific competition, food consumption by perch and ruffe decreased significantly when the complex substrate was used. With interspecific competition, food consumption by perch and ruffe did not change with substrate complexity, but perch clearly out‐competed ruffe on both substrates. Zebra mussel beds provide a refuge for macrozoobenthos against predation by ruffe and probably also by perch. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Parasite-mediated predation between native and invasive amphipods.

Parasites can structure biological communities directly through population regulation and indirectly by processes such as apparent competition. However, the role of parasites in the process of biological invasion is less well understood and mechanisms of parasite mediation of predation among hosts are unclear. Mutual predation between native and invading species is an important factor in determining the outcome of invasions in freshwater amphipod communities. Here, we show that parasites mediate mutual intraguild predation among native and invading species and may thereby facilitate the invasion process. We find that the native amphipod Gammarus duebeni celticus is host to a microsporidian parasite, Pleistophora sp. (new species), with a frequency of infection of 0-90%. However, the parasite does not infect three invading species, G. tigrinus, G. pulex and Crangonyx pseudogracilis. In field and laboratory manipulations, we show that the parasite exhibits cryptic virulence: the parasite does not affect host fitness in single-species populations, but virulence becomes apparent when the native and invading species interact. That is, infection has no direct effect on G. d. celticus survivorship, size or fecundity; however, in mixed-species experiments, parasitized natives show a reduced capacity to prey on the smaller invading species and are more likely to be preyed upon by the largest invading species. Thus, by altering dominance relationships and hierarchies of mutual predation, parasitism strongly influences, and has the potential to change, the outcome of biological invasions.

Twenty years of stand development in pure and mixed stands of Eucalyptus saligna and nitrogen-fixing Facaltaria moluccana

Abstract The vast majority of tropical tree plantations are monocultures, and only a few case studies have examined growth of mixed-species plantations. Mixtures that contain nitrogen (N)-fixing species may have the potential to increase overall plantation yields, but too few case studies are available to provide general insights. We extended previous measurements of 10 years of stand development in a mixed-species experiment in Hawaii through age 20 years. The plots had a constant total number of stems initially (2500 ha−1), but varying ratios of Eucalyptus saligna (Sm.) to nitrogen-fixing Facaltaria moluccana (Miquel) Barneby & Grimes (=Albizia Facaltaria (L.) Fosberg, and Paraserianthes Facaltaria (L.) I. Nielsen). Eucalyptus stem growth declined strongly in monocultures after age of 7 years, but showed no decline in mixtures. After 10 years of stand development, Eucalyptus stem mass was greatest in the monoculture, averaging about 155 Mg/ha (versus 140 Mg/ha in the 0.5 Facaltaria treatment); total (combined) stem mass for both species was almost twice as great in mixtures as in monocultures. By the age of 20 years, Eucalyptus stem mass was within a 20% range across treatments (260 Mg/ha), and total stem mass ranged up to 430 Mg/ha in the 0.75 Facaltaria treatment. Losses of stem mass from mortality were moderate for Eucalyptus between 18 and 20 years, averaging 20–25% of gross stem growth in mixed-species treatments, but 60% in the Eucalyptus monoculture. Mortality losses of stem mass were extremely high for Facaltaria in all plots, matching or exceeding gross stem growth from age 18 to 20 years. The size of individual Eucalyptus trees was much greater in the mixed plots than in the monoculture plots, so any short-term economic gain from the mixed-species plots may derive from larger tree sizes rather than from more wood mass per hectare. The longer term benefits of mixed plots may include the effects of Facaltaria on soil fertility and community diversity. Overall, these very large changes in ecosystem production in mixtures underscores the need for a large number of additional studies to examine other species combinations, sites, and silvicultural systems.

EXPLOITATIVE COMPETITION IN DIFFERENTLY SIZEDDAPHNIASPECIES: A MECHANISTIC EXPLANATION

The concept of the threshold food concentration that allows metabolic main- tenance of an animal (CO) has been used as an analogy to Tilman's R* (equilibrium resource requirements) to explain competitive abilities of cladoceran species. Until now, however, theoretical analysis has been hampered because CO was measured in experiments where cladoceran growth was determined in response to fixed, low food concentrations, not as a result of the cladocerans' grazing activity. CO is an indirect estimate from nonequilibrium conditions. It still needs to be shown that growing Daphnia are efficient enough to suppress resource concentrations to levels equivalent to their CO. We designed flow-through exper- iments to establish equilibrium conditions between algal inflow and cladoceran population biomass with three differently sized Daphnia species. The equilibrium food concentration resulting from the daphniids' grazing activity was labeled C*, as it can be used to predict competitive abilities in analogy to Tilman's R*. Equilibrium was reached in each experiment. Daphnia grew to carrying capacity without fluctuations in numbers or biomass, and biomass remained constant after this level was reached. Mortality was compensated for by somatic growth or, in the case of the small species D. ambigua, by production of offspring. When the carrying capacity was reached, algal concentrations remained constant at C*. As pre- dicted from previous growth experiments, larger species suppressed algal concentrations to lower levels, i.e., they were predicted to have higher competitive ability. This prediction was tested in a mixed-species experiment with D. pulicaria and the smaller D. galeata. That experiment clearly demonstrated the mechanism of exploitative competition. In the beginning, the smaller D. galeata performed better, but as soon as the food concentration fell below D. galeata's individually determined C*, the smaller species suffered mortality. At this point, D. pulicaria had not yet reached its C*. It continued to grow and replaced D. galeata. Numerical values of D. pulicaria's C* were slightly higher than the indepen- dently determined, corresponding CO values. This was to be expected as CO applies to zero mortality. However, the rank order among species was identical for both characteristics. We propose that both C* and CO have strong predictive power for mechanistic models of competitive interactions.

Competition between two aquatic detritivorous isopods : a laboratory study

The role of adult faeces in juvenile nutrition of two isopod species, Proasellus coxalis s.l. and Asellus aquaticus (L.), with similar trophic strategies and different reproductive output, has been studied in laboratory. Our aim was to consider the possible competitive mechanisms occurring at the beginning of the species coexistence using allopatric populations in single and mixed species experiments. Two series of competition experiments were performed. In the first, adult specimens were used for breeding and feeding trials. Both population dynamics and the percentage of ovigerous females and juveniles were evaluated during 10 months. Adult densities and juvenile percentage of A.aquaticus were lower in the presence of P. coxalis s.l. than when alone. At the end of the breeding experiments the dietary preferences of adults on a set of fungally conditioned leaf discs were not different among treatments. In the second series of experiments, the influence of coexistence on the feeding rates of young asellids and the relative importance of faeces and decaying plant material in their diet were investigated. Individual consumption by wild juveniles in multiple-choice laboratory experiments was measured by radioisotopes (322P). Juveniles of P. coxalis s.l. showed the highest ingestion rates. In co-occurrence, contrary to A.aquaticus, they were able to further increase feeding on parental faeces. The role of parental faeces in the diet of the two species juveniles and the competitive dominance of P. coxalis s.l. are discussed.

32Phosphorus transfer in systems of fish and amphipods exploiting detritus particles

Detrital size preferences of the fish Mugil sp. and the amphipod Echinogammarus veneris were determined in the laboratory by 32P. Three different size-classes of leaf-detritus particles, inoculated with a fungus, were offered separately in 24-h experiments. Detritus 32P increased with particle size, depending on the growth of microflora. Both species preferred the largest size-class. In mixed-species experiments, 32P uptake by Mugil was significantly less than when it was alone. A similar 32P transfer was measured in Mugil fed on the largest particles, previously utilized by E. veneris. This indicates that the feeding activity of the amphipod indirectly affects the detrital utilization by Mugil sp. It is suggested that the influence might depend on the microbial impoverishment and the change in the particle size.

Modification of sediment permeability and shear strength by two burrowing invertebrates

Laboratory experiments have been conducted on the effects of bioturbation by the amphipod Corophium volutator (Pallas) and the polychaete Nereis diversicolor (O.F. Muller) on the physical properties of estuarine muddy sand. Invertebrates and muddy sediment were collected from Langbank, Firth of Clyde, Scotland. Sediment columns were seeded with low, medium and high densities of invertebrates in single-and mixed-species experiments. Permeability decreased with increasing density of C. volutator, but increased with increasing density of N. diversicolor. Water content decreased with increasing density of both species. Shear strength increased with increasing density of both species, but more so with C. volutator than with N. diversicolor. Mixed-species effects on shear strength were additive, but some of the permeabilities were lower than predicted. Bivariate and multivariate correlation, and partial correlation analyses were applied to the data. Shear strength was negatively correlated with water content. The correlations between shear strength and permeability and between permeability and water content were affected differently by the two species. There were few partial correlations between the variables.

Transferrin: physiology and function in iron transport.

Since its identification about 30 years ago transferrin has attracted the interest of many investigators concerned with its structure, metal-binding properties, genetic polymorphism, and especially its role in the transport of iron within the body. Transferrin's two iron-binding sites appear to be structurally identical with equivalent iron binding after addition of iron in vitro. However, since the experiments of Fletcher and Huehns, the functional homogeneity of transferrin-bound iron has been questioned. Understanding of the precise mechanism of iron release from transferrin to receptor sites on reticulocytes and other tissues active in iron exchange is incomplete. Considerable evidence has been assembled from rats to support the Fletcher-Huehns hypothesis of selective release of A-site iron to erythrocyte precursors and placenta while B-site iron is delivered to hepatocytes. Results of experiments in rabbit and human systems remain controversial. Reasons for this controversy include: variations in the technique of iron addition to transferrin with the possibility of non-specific binding; variations in reticulocytes used in preparing selectively labelled transferrin and in its biological assay; and artifacts introduced in mixed-species experiments. Until methods are more refined and the transferrin-iron receptor interaction is better understood, the controversy about transferrins's iron transport function will persist.