Communality in microbial stress response and differential metabolic interactions revealed by time-series analysis of sponge symbionts

The diversity and function of sponge-associated symbionts is now increasingly understood; however, we lack an understanding of how they dynamically behave to ensure holobiont stability in the face of environmental variation. Here, we performed a metatransciptomic analysis on three microbial symbionts of the sponge Cymbastela concentrica in situ over 14 months and through differential gene expression and correlation analysis to environmental variables uncovered differences that speak to their metabolic activities and level of symbiotic and environmental interactions. The nitrite-oxidizing Ca. Porinitrospira cymbastela maintained a seemingly stable metabolism, with the few differentially expressed genes related only to stress responses. The heterotrophic Ca. Porivivens multivorans displayed differential use of holobiont-derived compounds and respiration modes, while the ammonium-oxidizing archaeon Ca. Nitrosopumilus cymbastelus differentially expressed genes related to phosphate metabolism and symbiosis effectors. One striking similarity between the symbionts was their similar variation in expression of stress-related genes. Our time-series study showed that the microbial community of C. concentrica undertakes dynamic gene expression adjustments in response to the surroundings, tuned to deal with general stress and metabolic interactions between holobiont members. The success of these dynamic adjustments likely underpins the stability of the sponge holobiont and may provide resilience against environmental change.     

The role of intracellular symbiotic bacteria in the amino acid nutrition of embryos from the black bean aphid,Aphis fabae

Aphids are well‐known for their symbiotic relationship with intracellular bacteria of the genus Buchnera (γ‐Proteobacteria). The symbiosis has a nutritional basis in that the bacteria supplement the aphid diet of phloem sap through the provision of essential amino acids. To date, few studies have considered the spatial complexity of the association, particularly the delineation of the symbiosis into embryo and maternal compartments. Here, we generate aposymbiotic (bacteria‐free) embryos of the black bean aphid, Aphis fabae (Scopoli) (Hemiptera: Aphididae), as our experimental model and demonstrate that embryos reared in culture media require an external supply of essential amino acids. Analysis of individual amino acid deletions from the culture medium indicate that the key individual amino acids for embryo growth are phenylalanine and valine derived from the maternal tissues, and tryptophan derived from Buchnera. These results are discussed in relation to our current limited understanding of nutrient supply to aphid embryos.     

Comparative gene expression in the symbiotic and aposymbiotic Aiptasia pulchella by expressed sequence tag analysis

Intracellular symbiotic relationships are prevalent between cnidarians, such as corals and sea anemones, and the photosynthetic dinoflagellate symbionts. However, there is little understanding about how the genes express when the symbiotic relationship is set up. To characterize genes involved in this association, the endosymbiosis between sea anemone, Aiptasia pulchella, and dinoflagellate zooxanthellae, Symbiodinium spp., was employed as a model. Two complementary DNA (cDNA) libraries were constructed from RNA isolated from symbiotic and aposymbiotic A. pulchella. Using single-pass sequencing of cDNA clones, a total of 870 expressed sequence tags (ESTs) clones were generated from the two libraries: 474 from symbiotic animal and 396 from aposymbiotic animal. The initial ESTs consisted of 143 clusters and 231 singletons. A BLASTX search revealed that 147 unique genes had similarities with protein sequences available from databases; 120 of these clones were categorized according to their putative function. However, many ESTs could not assign functionally. The putative roles of some of the identified genes relative to endosymbiosis were discussed. This is the first report of the use of EST analysis to examine the gene expression in symbiotic and aposymbiotic states of the cnidarians. The systematic analysis of EST from this study provides a useful database for future investigations of the molecular mechanisms involved in algal-cnidarian symbiosis.     

The elimination of intracellular microorganisms from insects: an analysis of antibiotic-treatment in the pea aphid (Acyrthosiphon pisum)

Antibiotics are routinely used to eliminate intracellular prokaryotic microorganisms from a wide range of insect species, but concerns about deleterious effects of antibiotic therapy on the insect host are seldom addressed. Here, the impact of antibiotic therapy in the symbiosis between the pea aphid Acyrthosiphon pisum and bacteria of the genus Buchnera is reviewed. Antibiotic-treatment produces aposymbiotic (i.e. symbiont-free) aphids, but does not depress the mitochondrial complement, the assimilation of dietary amino acids or the incorporation of amino acids into protein in these insects and does not impair osmoregulation, feeding rate and the capacity to penetrate plant tissues. It is concluded that the general malaise associated with aposymbiotic aphids is not attributable to a direct effect of the antibiotic. However, an important implication of this study is that aposymbiotic insects exhibit substantial metabolic adjustments to loss of the symbiosis; they are not simply aphids from which the symbiotic bacteria have been removed.     

Diatom invasion in the antarctic hexactinellid sponge Scolymastra joubini

Sponges often host large amounts of symbionts, mainly represented by cyanobacteria and dinoflagellates. Recent findings show a widespread presence of symbiotic diatoms living inside antarctic demosponges. In this paper, the invasion by large populations of the diatom Melosira sp. into specimens of the hexactinellid sponge Scolymastra joubini is reported. SEM analyses support the hypothesis that the embedded living diatoms have a negative impact on sponge tissues, leading to degenerative processes. Received: 1 December 1999 / Accepted: 20 February 2000     

Are Niemann‐Pick type C proteins key players in cnidarian–dinoflagellate endosymbioses?

The symbiotic interaction between cnidarians, such as corals and sea anemones, and the unicellular algae Symbiodinium is regulated by yet poorly understood cellular mechanisms, despite the ecological importance of coral reefs. These mechanisms, including host–symbiont recognition and metabolic exchange, control symbiosis stability under normal conditions, but also lead to symbiosis breakdown (bleaching) during stress. This study describes the repertoire of the sterol‐trafficking proteins Niemann‐Pick type C (NPC1 and NPC2) in the symbiotic sea anemone Anemonia viridis. We found one NPC1 gene in contrast to the two genes (NPC1 and NPC1L1) present in vertebrate genomes. While only one NPC2 gene is present in many metazoans, this gene has been duplicated in cnidarians, and we detected four NPC2 genes in A. viridis. However, only one gene (AvNPC2‐d) was upregulated in symbiotic relative to aposymbiotic sea anemones and displayed higher expression in the gastrodermis (symbiont‐containing tissue) than in the epidermis. We performed immunolabelling experiments on tentacle cross sections and demonstrated that the AvNPC2‐d protein was closely associated with symbiosomes. In addition, AvNPC1 and AvNPC2‐d gene expression was strongly downregulated during stress. These data suggest that AvNPC2‐d is involved in both the stability and dysfunction of cnidarian–dinoflagellate symbioses.     

Injection of essential amino acids substitutes for bacterial supply in aposymbiotic pea aphids (Acyrthosiphon pisum)

The symbiotic bacteria Buchnera contribute to the nutrition of pea aphids, Acyrthosiphon pisum, through the provision of essential amino acids which are lacking in the diet. However, chemically defined diets, containing nutritionally adequate amounts of essential amino acids, fail to rescue aposymbiotic aphids, in which the bacteria have been disrupted with antibiotics. In this study the injection of a mixture of essential amino acids into the haemocoel of aposymbiotic aphids was shown to alleviate, at least partially, the impact of symbiont loss. Specifically, the total amino acid content in the tissues of aposymbiotic aphids was reduced by approximately 40% to levels comparable with symbiotic insects, and there was a 1.7-fold increase in the number of embryos, suggesting that the availability of essential amino acids promotes aphid protein synthesis by rejuvenating the free amino acid pool of aposymbiotic aphids. In addition, a similar effect on the total amino acid content was observed when phenylalanine alone, but not glutamine, lysine or tryptophan, was injected into the haemocoel of aposymbiotic aphids, and there was also a significant increase in the number of embryos following injection of phenylalanine or tryptophan alone. The impact of amino acid injection on the embryo complement of aposymbiotic aphids was limited to an increase in the number of embryos, with no increase in basal embryo size. It is proposed that older embryos may rely on their own complement of symbiotic bacteria for essential amino acid provisioning. Taken together, the data highlight the importance of bacterial provisioning of essential amino acids, particularly the aromatic amino acids, in the intact symbiosis.     

On the functional significance of symbiotic microorganisms in the Homoptera: a comparative study of Acyrthosiphon pisum and Nilaparvata lugens

All phloem‐feeding Homoptera possess symbiotic microorganisms. Although the phylogenetic position and anatomical location of the micro‐ organisms differ, the underlying theme of the symbiosis is the same; the microorganisms improve the nutritional quality of the diet through the provision of essential amino acids. The symbiosis has been well documented in aphids, but little information is available from other homopteran groups. The impact of the loss of bacterial symbionts in the pea aphid Acyrthosiphon pisum Harris and eukaryotic yeast‐like symbionts in the Asian rice brown planthopper Nilaparvata lugens Stål was examined in parallel. The weight and relative growth rate of aphids and planthoppers was significantly reduced by symbiont loss, and characteristic features of aposymbiotic pea aphids, so‐called ‘metabolic signatures’, were, for the first time, observed in aposymbiotic N. lugens. For example, the amount of protein per unit fresh weight was reduced by 26 and 10%, and the free amino acid levels increased 1.8‐ and 1.4‐fold, in aposymbiotic A. pisum and N. lugens, respectively. In addition, the concentration of the amino acid glutamine was elevated in the tissues of aposymbiotic insects. The data are discussed in the context of our current understanding of the nutritional role of the symbiosis and the mechanisms of nitrogen metabolism in the two insect species. It is concluded that the metabolic adjustments of the insects to symbiont loss are broadly equivalent.     

Genetics of the relationship between the ciliate Paramecium bursaria and its symbiotic algae

Abstract. Paramecium bursaria , a freshwater protozoan, typically harbors hundreds of symbiotic algae ( Chlorella sp.) in its cytoplasm. The relationship between host paramecia and symbiotic algae is stable and mutually beneficial in natural environments. We recently collected an aposymbiotic strain of P. bursaria . Infection experiments revealed that the natural aposymbiotic strain (Ysa2) showed unstable symbiosis with Chlorella sp. The algae aggregated at the posterior region of the host, resulting in aposymbiotic cell production after cell division. Cross-breeding analyses were performed to determine the heritability of the aposymbiotic condition. In crosses of Ysa2 with symbiotic strains of P. bursaria , F1 progeny were able to form stable symbioses with Chlorella sp. However, unstable symbiosis, resembling Ysa2 infection, occurred in some F2 progeny of sibling crosses between symbiotic F1 clones. Infection experiments using aposymbiotic F2 cells showed that these F2 subclones have limited ability to reestablish the symbiosis. These results indicate that the maintenance of stable symbiosis is genetically controlled and heritable, and that Ysa2 is a mutant lacking the mechanisms to establish stable symbiosis with Chlorella sp.     

Comparative Proteomics of Symbiotic and Aposymbiotic Juvenile Soft Corals

The symbiotic association between corals and photosynthetic unicellular algae is of great importance in coral reef ecosystems. The study of symbiotic relationships is multidisciplinary and involves research in phylogeny, physiology, biochemistry, and ecology. An intriguing phase in each symbiotic relationship is its initiation, in which the partners interact for the first time. The examination of this phase in coral–algae symbiosis from a molecular point of view is still at an early stage. In the present study we used 2-dimensional polyacrylamide gel electrophoresis to compare patterns of proteins synthesized in symbiotic and aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens. This is the first work to search for symbiosis-specific proteins during the natural onset of symbiosis in early host ontogeny. The protein profiles reveal changes in the host soft coral proteome through development, but surprisingly virtually no changes in the host proteome as a function of symbiotic state.     

Water availability alters the tri-trophic consequences of a plant-fungal symbiosis

Plant–microbe protection symbioses occur when a symbiont defends its host against enemies (e.g., insect herbivores); these interactions can have important influences on arthropod abundance and composition. Understanding factors that generate context-dependency in protection symbioses will improve predictions on when and where symbionts are most likely to affect the ecology and evolution of host species and their associated communities. Of particular relevance are changes in abiotic contexts that are projected to accompany global warming. For example, increased drought stress can enhance the benefits of fungal symbiosis in plants, which may have multi-trophic consequences for plant-associated arthropods. Here, we tracked colonization of fungal endophyte-symbiotic and aposymbiotic Poa autumnalis (autumn bluegrass) by Rhopalosiphum padi (bird-cherry-oat aphids) and their parasitoids (Aphelinus sp.) following manipulations of soil water levels. Endophyte symbiosis significantly reduced plant colonization by aphids. Under low water, symbiotic plants also supported a significantly higher proportion of aphids that were parasitized by Aphelinus and had higher above-ground biomass than aposymbiotic plants, but these endophyte-mediated effects disappeared under high water. Thus, the multi-trophic consequences of plant-endophyte symbiosis were contingent on the abiotic context, suggesting the potential for complex responses in the arthropod community under future climate shifts.     

Aposymbiotic Plesiastrea versipora continues to produce cell-signalling molecules that regulate the carbon metabolism of symbiotic algae

The scleractinian coral Plesiastrea versipora produces cell signals that regulate the carbon metabolism of its symbiotic algae. Host release factor (HRF) stimulates the release of photosynthate, and photosynthesis inhibiting factor (PIF) partially inhibits carbon fixation in freshly isolated symbiotic algae. Naturally occurring aposymbiotic specimens of P. versipora are rare in Port Jackson, Australia, but one that was collected contained HRF and PIF. Artificially produced aposymbiotic corals of P. versipora that had been kept in the dark for up to 23 months continued to produce both HRF and PIF in the absence of photosynthetically active algae. Aposymbiotic P. versipora from which most of the tissue had been removed, regenerated when they were kept in the dark and fed; the regenerated tissue also contained HRF and PIF. These results suggest that the presence of symbiotic algae is not required for the production of HRF and PIF in P. versipora. We suggest that these cell signals may have evolved in response to symbiosis with Symbiodinium sp. but are now always expressed in the coral P. versipora.     

Long-term laboratory culture of symbiotic coral juveniles and their use in eco-toxicological study

Reef-building (or hermatypic) corals live in mutualistic symbiosis with the dinoflagellates Symbiodinium spp. (Alveolata, Dinophyceae, Gymnodiniales), and contribute to the accretion of coral reefs. Due to the difficulty in culturing them in laboratories, these ecologically important cnidarians have not been characterized extensively in physiological, biochemical, molecular and toxicological experiments. The present study was conducted to develop a model symbiosis system for long-term experimental analyses of a symbiotic coral. Aposymbiotic (symbiont-free) juveniles of the hermatypic coral Acropora tenuis were infected with three Symbiodinium strains, and the resulting symbiotic corals were examined for growth and maintenance of the symbiosis for approx. three months. Of the tested Symbiodinium cell lines, CCMP2467 (clade A1) inhabited the host the most densely, and the population in hospite did not decline over the period of three months in laboratory culture. The CCMP2467-inhabited juveniles outgrew the populations infected with the other two strains and aposymbiotic specimens. The A. tenuis juveniles in symbiosis with CCMP2467 cells were used in eco-toxicological tests to study long-term effects of two commonly used biocides (tributyltin-chloride and diuron). Delay in growth was observed after exposing the symbiotic juveniles to the two chemicals for approx. 50 days at the nominal concentrations of 0.4 and 1 μg/L, respectively.