Conflicting selection alters the trajectory of molecular evolution in a tripartite bacteria–plasmid–phage interaction

Bacteria engage in a complex network of ecological interactions, which includes mobile genetic elements (MGEs) such as phages and plasmids. These elements play a key role in microbial communities as vectors of horizontal gene transfer but can also be important sources of selection for their bacterial hosts. In natural communities, bacteria are likely to encounter multiple MGEs simultaneously and conflicting selection among MGEs could alter the bacterial evolutionary response to each MGE. Here, we test the effect of interactions with multiple MGEs on bacterial molecular evolution in the tripartite interaction between the bacterium, Pseudomonas fluorescens, the lytic bacteriophage, SBW25φ2, and conjugative plasmid, pQBR103, using genome sequencing of experimentally evolved bacteria. We show that individually, both plasmids and phages impose selection leading to bacterial evolutionary responses that are distinct from bacterial populations evolving without MGEs, but that together, plasmids and phages impose conflicting selection on bacteria, constraining the evolutionary responses observed in pairwise interactions. Our findings highlight the likely difficulties of predicting evolutionary responses to multiple selective pressures from the observed evolutionary responses to each selective pressure alone. Understanding evolution in complex microbial communities comprising many species and MGEs will require that we go beyond studies of pairwise interactions.     

Use of carbon monoxide and hydrogen by a bacteria–animal symbiosis from seagrass sediments

The gutless marine worm Olavius algarvensis lives in symbiosis with chemosynthetic bacteria that provide nutrition by fixing carbon dioxide (CO₂) into biomass using reduced sulfur compounds as energy sources. A recent metaproteomic analysis of the O. algarvensis symbiosis indicated that carbon monoxide (CO) and hydrogen (H₂) might also be used as energy sources. We provide direct evidence that the O. algarvensis symbiosis consumes CO and H₂. Single cell imaging using nanoscale secondary ion mass spectrometry revealed that one of the symbionts, the γ3‐symbiont, uses the energy from CO oxidation to fix CO₂. Pore water analysis revealed considerable in‐situ concentrations of CO and H₂ in the O. algarvensis environment, Mediterranean seagrass sediments. Pore water H₂ concentrations (89–2147 nM) were up to two orders of magnitude higher than in seawater, and up to 36‐fold higher than previously known from shallow‐water marine sediments. Pore water CO concentrations (17–51 nM) were twice as high as in the overlying seawater (no literature data from other shallow‐water sediments are available for comparison). Ex‐situ incubation experiments showed that dead seagrass rhizomes produced large amounts of CO. CO production from decaying plant material could thus be a significant energy source for microbial primary production in seagrass sediments.     

Acidic duodenal pH alters gene expression in the cystic fibrosis mouse pancreas

The duodenum is abnormally acidic in cystic fibrosis (CF) due to decreased bicarbonate ion secretion that is dependent on the CF gene product CFTR. In the CFTR null mouse, the acidic duodenum results in increased signaling from the intestine to the exocrine pancreas in an attempt to stimulate pancreatic bicarbonate ion secretion. Excess stimulation is proposed to add to the stress/inflammation of the pancreas in CF. DNA microarray analysis of the CF mouse revealed altered pancreatic gene expression characteristic of stress/inflammation. When the duodenal pH was corrected genetically (crossing CFTR null with gastrin null mice) or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalized. It is concluded that the acidic duodenal pH in CF contributes to the stress on the exocrine pancreas and that normalizing duodenal pH reduces this stress.     

Manifold aspects of specificity in a nematode–bacterium mutualism

Coevolution in mutualistic symbiosis can yield, because the interacting partners share common interests, to coadaptation: hosts perform better when associated with symbionts of their own locality than with others coming from more distant places. However, as the two partners of a symbiosis might also experience conflicts over part of their life cycle, coadaptation might not occur for all life‐history traits. We investigated this issue in symbiotic systems where nematodes (Steinernema) and bacteria (Xenorhabdus) reproduce in insects they have both contributed to kill. Newborn infective juveniles (IJs) that carry bacteria in their intestine then disperse from the insect cadaver in search of a new host to infect. We ran experiments where nematodes coinfect insects with bacteria that differ from their native symbiont. In both Steinernema carpocapsae/Xenorhabdus nematophila and Steinernema feltiae/Xenorhabdus bovienii symbioses, we detected an overall specificity which favours the hypothesis of a fine‐tuned co‐adaptation process. However, we also found that the life‐history traits involved in specificity strongly differ between the two model systems: when associated with strains that differ too much from their native symbionts, S. carpocapsae has low parasitic success, whereas S. feltiae has low survival in dispersal stage.     

Extreme ecological stoichiometry of a bark beetle–fungus mutualism

1. Ecological stoichiometry theory was applied to investigate how a consumer contends with an extreme elemental mismatch between its food and its body via symbiotic facilitation. 2. The beetle Dendroctonus brevicomis LeConte develops in bark, a substrate extremely low in nitrogen (N) and phosphorus (P). Its survival there depends on interactions with mutualist and antagonist fungi. 3. This study found that mutualists transfer N and P from sapwood and phloem into bark, where beetles feed, whereas the antagonist moves these elements only to phloem, resulting in starvation of the insect. However, even with mutualists, N and P concentrations remained low in bark, resulting in low N and extremely low P concentrations in the beetle. 4. The N:P ratios found in D. brevicomis larvae were the highest thus far reported for beetles and among the highest for insects and invertebrates. This suggests that the beetle has evolved additional, nutrient‐sparing adaptations.     

Herbivory in a spider through exploitation of an ant–plant mutualism

Spiders are thought to be strict predators [1]. We describe a novel exception: Bagheera kiplingi, a Neotropical jumping spider (Salticidae) that exploits a well-studied ant–plant mutualism, is predominantly herbivorous. From behavioral field observations and stable-isotope analyses, we show that the main diet of this host-specific spider comprises specialized leaf tips (Beltian food bodies; Figure 1A) from Vachellia spp. ant-acacias (formerly Acacia spp.), structures traded for protection in the plant's coevolved mutualism with Pseudomyrmex spp. ants that inhabit its hollow thorns [2]. This is the first report of a spider that feeds primarily and deliberately on plants.     

Plant glandular trichomes mediate protective mutualism in a spider–plant system

1. Although several species of Peucetia (Oxyopidae) live strictly in association with plants bearing glandular trichomes worldwide, to date little is known about whether these associations are mutualistic. 2. In this study we manipulated the presence of Peucetia flava on the glandular plant Rhynchanthera dichotoma in the rainy and post‐rain season, to test the strength of its effects on leaf, bud, and flower damage and plant reproductive output. In addition, we ran independent field experiments to examine whether these sticky structures improve spider fidelity to plants. 3. Peucetia suppressed some species of foliar phytophages, but not others. Although spiders have reduced levels of leaf herbivory, this phenomenon was temporally conditional, i.e. occurred only in the post‐rain but not in the rainy season. Floral herbivory was also reduced in the presence of spiders, but these predators did not affect plant fitness components. 4. Plants that had their glandular trichomes removed retained fewer insects than those bearing such structures. Spiders remained longer on plants with glandular trichomes than on plants in which these structures had been removed. Isotopic analyses showed that spiders that fed on live and dead labelled flies adhered to the glandular hairs in similar proportions. 5. Spiders incurred no costs to the plants, but can potentially increase individual plant fitness by reducing damage to reproductive tissues. Temporal conditionality probably occurred because plant productivity exceeded herbivore consumption, thus dampening top‐down effects. Specialisation to live on glandular plants may have favoured scavenging behaviour in Peucetia, possibly an adaptation to periods of food scarcity.     

Interaction of Melanin with Proteins – The Importance of an Acidic Intramelanosomal pH

Melanin is a highly irregular heteropolymer consisting of monomeric units derived from the enzymatic oxidation of the amino acid tyrosine. The process of melanin formation takes place in specialized acidic organelles (melanosomes) in melanocytes. The process of melanin polymerization requires an alkaline pH in vitro, and therefore, the purpose of an acidic environment in vivo remains a mystery. It is known that melanin is always bound to protein in vivo. It is also seen that polymerization in vitro at an acidic pH necessarily requires the presence of proteins. The effect of various model proteins on melanin synthesis and their interaction with melanin was studied. It was seen that many proteins could increase melanin synthesis at an acidic pH, and that different proteins resulted in the formation of different states of melanin, i.e., a precipitate or a soluble, protein‐bound form. We also present evidence to show that soluble protein‐bound melanin is present in vivo (in B16 cells as well as in B16 melanoma tissue). An acidic pH appeared to be necessary to ensure the formation of a uniform, very high molecular weight melano–protein complex. The interaction between melanin and proteins appears to be largely charge‐dependent as evidenced by zeta potential measurements, and this interaction is also increased in an acidic pH. Thus, it appears that an acidic intramelanosomal pH is essential to ensure maximum interaction between protein and melanin, and also to ensure that all the melanin formed is protein‐bound.     

Helicobacter pylori lipopolysaccharide modification, Lewis antigen expression, and gastric colonization are cholesterol-dependent

Background  

Helicobacter pylori specifically takes up cholesterol and incorporates it into the bacterial membrane, yet little is currently known about cholesterol's physiological roles. We compared phenotypes and in vivo colonization ability of H. pylori grown in a defined, serum-free growth medium, F12 with 1 mg/ml albumin containing 0 to 50 μg/ml cholesterol.     

Presence of weed fungus in a non‐social beetle–fungus cultivation mutualism

1. Farming by non‐human organisms has arisen independently in several animal lineages, allowing them to survive on food sources that are otherwise difficult to access. However, agricultural gardens are prone to invasion by parasites that overgrow cultivars in the absence of host animals. The presence of garden parasites and associated host adaptations are well studied in advanced fungal agriculture practised by social insects (ants, termites, and ambrosia beetles), but the impact of garden parasites in more primitive forms of agriculture is poorly known. The aim of the present study was to report the presence of weed fungi in fungal gardens of the non‐social lizard beetle Doubledaya bucculenta Lewis (Coleoptera: Erotylidae: Languriinae), which cultivates the yeast Wickerhamomyces anomalus (E.C. Hansen) Kurtzman, Robnett & Bas.‐Powers in the internodes of recently dead bamboo culms. 2. The filamentous fungi Arthrinium spp., Fusarium spp., and an unidentified species were isolated from the inner wall and insect‐made wall debris of bamboo internodes inhabited by the beetle. 3. When cultured together with the yeast in the absence of host larvae, Arthrinium sp.1 outcompeted the yeast, suggesting that this filamentous fungus can invade yeast gardens but is normally suppressed under natural conditions. 4. Rearing experiments showed that beetle larvae grew faster when grown on yeast cultures than when fed only Arthrinium sp.1. 5. These results suggest that Arthrinium sp.1 acts as a weed in the fungal gardens of D. bucculenta, inhibiting both growth of the beetle larvae and proliferation of the beneficial yeast.     

Infiltration of a facultative ant–plant mutualism by the introduced Argentine ant: effects on mutualist diversity and mutualism benefits

1. Ant–plant mutualisms have been the focus of considerable empirical research, but few studies have investigated how introduced ants affect these interactions. Using 2 years of survey data, this study examines how the introduced Argentine ant [Linepithema humile (Mayr)] differs from native ants with respect to its ability to protect the extrafloral nectary‐bearing coast barrel cactus (Ferocactus viridescens) in Southern California. 2. Eighteen native ant species visited cacti in uninvaded areas, but cacti in invaded areas were primarily visited by the Argentine ant. The main herbivore of the coast barrel cactus present at the study sites is a leaf‐footed bug (Narnia wilsoni). 3. Herbivore presence (the fraction of surveys in which leaf‐footed bugs were present on individual cacti) was negatively related to ant presence (the fraction of surveys in which ants were present on individual cacti). Compared with cacti in uninvaded areas, those in invaded areas were less likely to have herbivores and when they did had them less often. 4. Seed mass was negatively related to herbivore presence, and this relationship did not differ for cacti in invaded areas versus those in uninvaded areas. 5. Although the Argentine ant might provide superior protection from herbivores, invasion‐induced reductions in ant mutualist diversity could potentially compromise plant reproduction. The cumulative number of ant species on individual cacti over time was lower in invaded areas and was associated with a shortened seasonal duration of ant protection and reduced seed mass. These results support the hypothesis that multiple partners may enhance mutualism benefits.     

Parasites and mutualism function: measuring enemy‐free space in a fig–pollinator symbiosis

Mutualisms involve cooperation between species and underpin several ecosystem functions. However, there is also conflict between mutualists, because their interests are not perfectly aligned. In addition, most mutualisms are exploited by parasites. Here, we study the interplay between cooperation, conflict and parasitism in the mutualism between fig trees and their pollinator wasps. Conflict occurs because each fig ovary can nurture either one seed or one pollinator offspring and, while fig trees benefit directly from seeds and pollinator offspring (pollen vectors), pollinators only benefit directly from pollinator offspring. The mechanism(s) of conflict resolution is debated, but must explain the widespread observation that pollinators develop in inner, and seeds in outer, layers of fig flowers. We recently suggested a role for non‐pollinating figs wasps (NPFWs) that are natural enemies or competitors of the pollinators and lay their eggs through the fig wall. Most NPFW offspring develop in outer and middle layer flowers, suggesting that inner flowers provide enemy‐free space for pollinator offspring. Here, we test the hypothesis that NPFWs cannot reach inner flowers, by measuring wasp and fig morphology at the species‐specific times of NPFW attack in the field. We found that three species of Sycoscapter and Philotrypesis wasps that parasitise pollinators could reach 34–73%, 75–92% and 82–97% of fig ovaries, respectively. Meanwhile, Eukobelea and Pseudidarnes gall‐formers, despite having shorter ovipositors, can access almost all fig flowers (93–99% and 100%), because they attack smaller (younger) fig fruits. Our mechanistic results from ovipositing wasps support spatial patterns of wasp offspring segregation within figs to suggest that inner ovules provide enemy‐free‐space for pollinators. This may contribute to mutualism stability by helping select for pollinators to avoid laying eggs where they are likely to be parasitised. These outer flowers then remain free to develop as seeds, promoting mutualism persistence.     

Introduced ants reduce interaction diversity in a multi‐species,ant–aphid mutualism

Mutualisms contribute in fundamental ways to the origin, maintenance and organization of biological diversity. Introduced species commonly participate in mutualisms, but how this phenomenon affects patterns of interactions among native mutualists remains incompletely understood. Here we examine how networks of interactions among aphid‐tending ants, ant‐tended aphids, and aphid‐attacking parasitoid wasps differ between 12 spatially paired riparian study sites with and without the introduced Argentine ant Linepithema humile in southern California. To resolve challenges in species identification, we used DNA barcoding to identify aphids and screen for parasitoid wasps (developing inside their aphid hosts) from 170 aphid aggregations sampled on arroyo willow Salix lasiolepis. Compared to uninvaded sites, invaded sites supported significantly fewer species of aphid‐tending ants and ant‐tended aphids. At invaded sites, for example, we found only two species of ant‐tended aphids, which were exclusively tended by L. humile, whereas at uninvaded sites we found 20 unique ant–aphid interactions involving eight species of ant‐tended aphids and nine species of aphid‐tending ants. Ant–aphid linkage density was thus significantly lower at invaded sites compared to uninvaded sites. We detected aphid parasitoids in 14% (28/198) of all aphid aggregations. Although the level of parasitism did not differ between invaded and uninvaded sites, more species of wasps were detected within uninvaded sites compared to invaded sites. These results provide a striking example of how the assimilation of introduced species into multi‐species mutualisms can reduce interaction diversity with potential consequences for species persistence.     

Geographically structured genetic variation in the Medicago lupulina–Ensifer mutualism

Gene flow between genetically differentiated populations can maintain variation in species interactions, especially when population structure is congruent between interacting species. However, large‐scale empirical comparisons of the population structure of interacting species are rare, particularly in positive interspecific interactions (mutualisms). One agriculturally and ecologically important mutualism is the partnership between legume plants and rhizobia. Through characterizing and comparing the population genomic structure of the legume Medicago lupulina and two rhizobial species (Ensifer medicae and E. meliloti), we explored the spatial scale of population differentiation between interacting partners in their introduced range in North America. We found high proportions of E. meliloti in southeastern populations and high proportions of E. medicae in northwestern populations. Medicago lupulina and the Ensifer genus showed similar patterns of spatial genetic structure (isolation by distance). However, we detected no evidence of isolation by distance or population structure within either species of bacteria. Genome‐wide nucleotide diversity within each of the two Ensifer species was low, suggesting limited introduction of strains, founder events, or severe bottlenecks. Our results suggest that there is potential for geographically structured coevolution between M. lupulina and the Ensifer genus, but not between M. lupulina and either Ensifer species.     

Previously unrecognized stages of species‐specific colonization in the mutualism between Xenorhabdus bacteria and Steinernema nematodes

The specificity of a horizontally transmitted microbial symbiosis is often defined by molecular communication between host and microbe during initial engagement, which can occur in discrete stages. In the symbiosis between Steinernema nematodes and Xenorhabdus bacteria, previous investigations focused on bacterial colonization of the intestinal lumen (receptacle) of the nematode infective juvenile (IJ), as this was the only known persistent, intimate and species‐specific contact between the two. Here we show that bacteria colonize the anterior intestinal cells of other nematode developmental stages in a species‐specific manner. Also, we describe three processes that only occur in juveniles that are destined to become IJs. First, a few bacterial cells colonize the nematode pharyngeal‐intestinal valve (PIV) anterior to the intestinal epithelium. Second, the nematode intestine constricts while bacteria initially remain in the PIV. Third, anterior intestinal constriction relaxes and colonizing bacteria occupy the receptacle. At each stage, colonization requires X. nematophila symbiosis region 1 (SR1) genes and is species‐specific: X. szentirmaii, which naturally lacks SR1, does not colonize unless SR1 is ectopically expressed. These findings reveal new aspects of Xenorhabdus bacteria interactions with and transmission by theirSteinernema nematode hosts, and demonstrate that bacterial SR1 genes aid in colonizing nematode epithelial surfaces.     

Cascading trait‐mediation: disruption of a trait‐mediated mutualism by parasite‐induced behavioral modification

Trait‐mediated indirect interactions (TMII) are important driving‐forces causing trophic cascades in aquatic and terrestrial food webs. Furthermore, since most biological communities are not simple food chains but complex networks of interactions, one TMII within a community might easily be influenced by another TMII. In other words, TMII themselves can be cascades with potential implications for community dynamics. Here we report on one of such cascade, where a parasitic fly induces behavioral changes that disrupt a trait‐mediated ant–hemipteran mutualism. We show that during parasite‐induced low‐activity periods, the ant Azteca instabilis fails to protect its mutualistic scale‐insect partner against predatory ladybeetles. Thus, in the presence of the parasite, ladybeetles ate as many scales in ant‐patrolled plants as they did in ant‐free plants. These results demonstrate how, through a cascade of trait‐mediated interactions, associations between members of a community can be drastically altered.     

Context‐dependency of a complex fruit–frugivore mutualism: temporal variation in crop size and neighborhood effects

The quantity of fruit consumed by dispersers is highly variable among individuals within plant populations. The outcome of such selection operated by frugivores has been examined mostly with respect to changing spatial contexts. The influence of varying temporal contexts on frugivore choice, and their possible demographic and evolutionary consequences is poorly understood. We examined if temporal variation in fruit availability across a hierarchy of nested temporal levels (interannual, intraseasonal, 120 h, 24 h) altered frugivore choice for a complex seed dispersal system in dry tropical forests of southern India. The interactions between Phyllanthus emblica and its primary disperser (ruminants) was mediated by another frugivore (a primate), which made large quantities of fruit available on the ground to ruminants. The direction and strength of crop size and neighborhood effects on this interaction varied with changing temporal contexts. Fruit availability was higher in the first of the two study years, and at the start of the season in both years. Fruit persistence on trees, determined by primate foraging, was influenced by crop size and conspecific neighborhood densities only in the high fruit availability year. Fruit removal by ruminants was influenced by crop size in both years and neighborhood densities only in the high availability year. In both years, these effects were stronger at the start of the season. Intraseasonal reduction in fruit availability diminished inequalities in fruit removal by ruminants and the influence of crop size and fruiting neighborhoods. All trees were not equally attractive to frugivores in a P. emblica population at all points of time. Temporal asymmetry in frugivore‐mediated selection could reduce potential for co‐evolution between frugivores and plants by diluting selective pressures. Inter‐dependencies formed between disparate animal consumers can add additional levels of complexity to plant–frugivore mutualistic networks and have potential reproductive consequences for specific individuals within populations.     

Acidic amino acid transport in animal cells and tissues

1. The occurrence and characterization of acidic amino acid transport in the plasma membrane of a variety of cells and tissues of a number of organisms is reviewed. 2. Several cell types, especially in brain, possess both high- and low-affinity transport systems for acidic amino acids. 3. High-affinity systems in brain may function to remove neurotransmitter amino acid from the extracellular environment. 4. Many cell systems for acidic amino acid transport are energized by an inwardly directed Na+ gradient. Moreover, certain cell types, such as rat brain neurons, human placental trophoblast and rabbit and rat kidney cortex epithelium, respond to an outwardly directed K+ gradient as an additional source of energization. This simultaneous action may account for the high accumulation ratios seen with acidic amino acids. 5. Rabbit kidney has been found to have a glutamate-H+ co-transport system which is subject to stimulation by protons in the medium. 6. Acidic amino acid transport in rat brain neurons occurs with a stoichiometric coupling of 1 mol of amino acid to 2 mol of Na+. For rabbit intestine, one Na+ is predicted to migrate for each mol of amino acid. 7. Uptake in rat kidney cortex and in high-K+ dog erythrocytes is electrogenic. However, uptake in rabbit and newt kidney and in rat and rabbit intestine is electroneutral. 8. Na+-independent acidic amino acid transport systems have been described in the mouse lymphocyte, the human fibroblast, the mouse Ehrlich cell and in rat hepatoma cells. 9. In a number of cell systems, D-acidic amino acids have substantial affinity for transport; D-glutamate, in a number of systems, however, appears to have little reactivity. 10. Acidic amino acid transport in some cell systems appears to occur via the "classical" routes (Christensen, Adv. Enzymol. Relat. Areas Mol. Biol. 49, 41-101, 1979). For example, uptake in the Ehrlich cell is partitioned between the Na+-dependent A system (which transports a wide spectrum of neutral amino acids), the Na+-dependent ASC system (which transports alanine, serine, threonine, homoserine, etc.), and the Na+-independent L system (which shows reactivity centering around neutral amino acids such as leucine and phenylalanine). Also, a minor component of uptake in mouse lymphocytes occurs by a route resembling the A system. 11. Human fibroblasts possess a Na+-independent adaptive transport system for cystine and glutamate that is enhanced in activity by cystine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)