Extinction of anciently associated gut bacterial symbionts in a clade of stingless bees

Animal-microbe symbioses are often stable for millions of years. An example is the clade consisting of social corbiculate bees—honeybees, bumblebees, and stingless bees—in which a shared ancestor acquired specialized gut bacteria that subsequently diversified with hosts. This model may be incomplete, however, as few microbiomes have been characterized for stingless bees, which are diverse and ecologically dominant pollinators in the tropics. We surveyed gut microbiomes of Brazilian stingless bees, focusing on the genus Melipona, for which we sampled multiple species and biomes. Strikingly, Melipona lacks Snodgrassella and Gilliamella, bacterial symbionts ubiquitous in other social corbiculate bees. Instead, Melipona species harbor more environmental bacteria and bee-specific Starmerella yeasts. Loss of Snodgrassella and Gilliamella may stem from ecological shifts in Melipona or the acquisition of new symbionts as functional replacements. Our findings demonstrate the value of broadly sampling microbiome biodiversity and show that even ancient symbioses can be lost.Subject terms: Symbiosis, Microbiome, Microbial ecology, Metagenomics, Microbial ecology     

Colony contact contributes to the diversity of gut bacteria in bumblebees (Bombus terrestris)

Social bees, like honeybees and bumblebees, have a close contact with nest mates of different developmental stages and generations. This could enhance bacterial transfer between nest mates and offers opportunities for direct transfer of symbionts from one generation to the next, resulting in a stable host specific gut microbiota. Gut symbionts of honeybees and bumblebees have been suggested to contribute in digestion and protection against parasites and pathogens. Here we studied the impact of contact with the bumblebee colony on the colonization potential of the bacterial families (i.e., Neisseriaceae, Orbaceae, Lactobacillaceae and Bifidobacteriaceae) occurring in the gut of adult bumblebees (Bombus terrestris). Bacterial profiles of the gut microbiota of B. terrestris were determined based on the hypervariable V4 region of the 16S rRNA using paired‐end Illumina sequencing. In our experiments, we created different groups in which we gradually reduced the contact with nest mates and hive material. We made 3 observations: (i) reducing the contact between the colony and the bumblebee during adult life resulted in a significant drop in the relative abundance of Lactobacillus bombicola and Lactobacillus bombi; (ii) Bifidobacteriaceae required contact with nest mates to colonize the gut of B. terrestris and a significant lower bacterial diversity was observed in bumblebees that were completely excluded from colony contact during the adult life; (iii) Snodgrassella and Gilliamella were able to colonize the gut of the adult bumblebee without any direct contact with nest mates in the adult life stage. These results indicate the impact of the colony life on the diversity of the characteristic bumblebee gut bacteria.     

Individual variation in growth and physiology of symbionts in response to temperature

In many cases, understanding species’ responses to climate change requires understanding variation among individuals in response to such change. For species with strong symbiotic relationships, such as many coral reef species, genetic variation in symbiont responses to temperature may affect the response to increased ocean temperatures. To assess variation among symbiont genotypes, we examined the population dynamics and physiological responses of genotypes of Breviolum antillogorgium in response to increased temperature. We found broad temperature tolerance across genotypes, with all genotypes showing positive growth at 26, 30, and 32°C. Genotypes differed in the magnitude of the response of growth rate and carrying capacity to increasing temperature, suggesting that natural selection could favor different genotypes at different temperatures. However, the historical temperature at which genotypes were reared (26 or 30°C) was not a good predictor of contemporary temperature response. We found increased photosynthetic rates and decreased respiration rates with increasing contemporary temperature, and differences in physiology among genotypes, but found no significant differences in the response of these traits to temperature among genotypes. In species with such broad thermal tolerance, selection experiments on symbionts outside of the host may not yield results sufficient for evolutionary rescue from climate change.     

Leptin Signaling Is Required for Adaptive Changes in Food Intake,but Not Energy Expenditure,in Response to Different Thermal Conditions

Survival of free-living animals depends on the ability to maintain core body temperature in the face of rapid and dramatic changes in their thermal environment. If food intake is not adjusted to meet the changing energy demands associated with changes of ambient temperature, a serious challenge to body energy stores can occur. To more fully understand the coupling of thermoregulation to energy homeostasis in normal animals and to investigate the role of the adipose hormone leptin to this process, comprehensive measures of energy homeostasis and core temperature were obtained in leptin-deficient ob/ob mice and their wild-type (WT) littermate controls when housed under cool (14°C), usual (22°C) or ∼ thermoneutral (30°C) conditions. Our findings extend previous evidence that WT mice robustly defend normothermia in response to either a lowering (14°C) or an increase (30°C) of ambient temperature without changes in body weight or body composition. In contrast, leptin-deficient, ob/ob mice fail to defend normothermia at ambient temperatures lower than thermoneutrality and exhibit marked losses of both body fat and lean mass when exposed to cooler environments (14°C). Our findings further demonstrate a strong inverse relationship between ambient temperature and energy expenditure in WT mice, a relationship that is preserved in ob/ob mice. However, thermal conductance analysis indicates defective heat retention in ob/ob mice, irrespective of temperature. While a negative relationship between ambient temperature and energy intake also exists in WT mice, this relationship is disrupted in ob/ob mice. Thus, to meet the thermoregulatory demands of different ambient temperatures, leptin signaling is required for adaptive changes in both energy intake and thermal conductance. A better understanding of the mechanisms coupling thermoregulation to energy homeostasis may lead to the development of new approaches for the treatment of obesity.     

Loss and resiliency of social amoeba symbiosis under simulated warming

Anthropogenic global change is increasingly raising concerns about collapses of symbiotic interactions worldwide. Therefore, understanding how climate change affects symbioses remains a challenge and demands more study. Here, we look at how simulated warming affects the social ameba Dictyostelium discoideum and its relationship with its facultative bacterial symbionts, Paraburkholderia hayleyella and Paraburkholderia agricolaris. We cured and cross‐infected ameba hosts with different symbionts. We found that warming significantly decreased D. discoideum''s fitness, and we found no sign of local adaptation in two wild populations. Experimental warming had complex effects on these symbioses with responses determined by both symbiont and host. Neither of these facultative symbionts increases its hosts’ thermal tolerance. The nearly obligate symbiont with a reduced genome, P. hayleyella, actually decreases D. discoideum''s thermal tolerance and even causes symbiosis breakdown. Our study shows how facultative symbioses may have complex responses to global change.     

Microbial Communities of Three Sympatric Australian Stingless Bee Species

Bacterial symbionts of insects have received increasing attention due to their prominent role in nutrient acquisition and defense. In social bees, symbiotic bacteria can maintain colony homeostasis and fitness, and the loss or alteration of the bacterial community may be associated with the ongoing bee decline observed worldwide. However, analyses of microbiota associated with bees have been largely confined to the social honeybees (Apis mellifera) and bumblebees (Bombus spec.), revealing – among other taxa – host-specific lactic acid bacteria (LAB, genus Lactobacillus) that are not found in solitary bees. Here, we characterized the microbiota of three Australian stingless bee species (Apidae: Meliponini) of two phylogenetically distant genera (Tetragonula and Austroplebeia). Besides common plant bacteria, we find LAB in all three species, showing that LAB are shared by honeybees, bumblebees and stingless bees across geographical regions. However, while LAB of the honeybee-associated Firm4–5 clusters were present in Tetragonula, they were lacking in Austroplebeia. Instead, we found a novel clade of likely host-specific LAB in all three Australian stingless bee species which forms a sister clade to a large cluster of Halictidae-associated lactobacilli. Our findings indicate both a phylogenetic and geographical signal of host-specific LAB in stingless bees and highlight stingless bees as an interesting group to investigate the evolutionary history of the bee-LAB association.     

Topographic and vegetation drivers of thermal heterogeneity along the boreal–grassland transition zone in western Canada: Implications for climate change refugia

Climate change refugia are areas that are relatively buffered from contemporary climate change and may be important safe havens for wildlife and plants under anthropogenic climate change. Topographic variation is an important driver of thermal heterogeneity, but it is limited in relatively flat landscapes, such as the boreal plain and prairie regions of western Canada. Topographic variation within this region is mostly restricted to river valleys and hill systems, and their effects on local climates are not well documented. We sought to quantify thermal heterogeneity as a function of topography and vegetation cover within major valleys and hill systems across the boreal–grassland transition zone.Using iButton data loggers, we monitored local temperature at four hills and 12 river valley systems that comprised a wide range of habitats and ecosystems in Alberta, Canada (N = 240), between 2014 and 2020. We then modeled monthly temperature by season as a function of topography and different vegetation cover types using general linear mixed effect models.Summer maximum temperatures (T _max) varied nearly 6°C across the elevation gradient sampled. Local summer mean (T _mean) and maximum (T _max) temperatures on steep, north‐facing slopes (i.e., low levels of potential solar radiation) were up to 0.70°C and 2.90°C cooler than highly exposed areas, respectively. T _max in incised valleys was between 0.26 and 0.28°C cooler than other landforms, whereas areas with greater terrain roughness experienced maximum temperatures that were up to 1.62°C cooler. We also found that forest cover buffered temperatures locally, with coniferous and mixedwood forests decreasing summer T _mean from 0.23 to 0.72°C and increasing winter T _min by up to 2°C, relative to non‐forested areas.Spatial predictions of temperatures from iButton data loggers were similar to a gridded climate product (ClimateNA), but the difference between them increased with potential solar radiation, vegetation cover, and terrain roughness.Species that can track their climate niche may be able to compensate for regional climate warming through local migrations to cooler microsites. Topographic and vegetation characteristics that are related to cooler local climates should be considered in the evaluation of future climate change impacts and to identify potential refugia from climate change.     

Removal of gut symbiotic bacteria negatively affects life history traits of the shield bug,Graphosoma lineatum

The shield bug, Graphosoma lineatum (Heteroptera, Pentatomidae), harbors extracellular Pantoea‐like symbiont in the enclosed crypts of the midgut. The symbiotic bacteria are essential for normal longevity and fecundity of this insect. In this study, life table analysis was used to assess the biological importance of the gut symbiont in G. lineatum. Considering vertical transmission of the bacterial symbiont through the egg surface contamination, we used surface sterilization of the eggs to remove the symbiont. The symbiont population was decreased in the newborn nymphs hatched from the surface‐sterilized eggs (the aposymbiotic insects), and this reduction imposed strongly negative effects on the insect host. We found significant differences in most life table parameters between the symbiotic insects and the aposymbiotics. The intrinsic rate of increase in the control insects (0.080 ± 0.003 day⁻¹) was higher than the aposymbiotic insects (0.045 ± 0.007 day⁻¹). Also, the net reproductive and gross reproductive rates were decreased in the aposymbiotic insects (i.e., 20.770 ± 8.992 and 65.649 ± 27.654 offspring/individual, respectively), compared with the symbiotic insects (i.e., 115.878 ± 21.624 and 165.692 ± 29.058 offspring/individual, respectively). These results clearly show biological importance of the symbiont in G. lineatum.     

Nitrate competition in a coral symbiosis varies with temperature among Symbiodinium clades

Many reef-building corals form symbioses with dinoflagellates from the diverse genus Symbiodinium. There is increasing evidence of functional significance to Symbiodinium diversity, which affects the coral holobiont''s response to changing environmental conditions. For example, corals hosting Symbiodinium from the clade D taxon exhibit greater resistance to heat-induced coral bleaching than conspecifics hosting the more common clade C. Yet, the relatively low prevalence of clade D suggests that this trait is not advantageous in non-stressful environments. Thus, clade D may only be able to out-compete other Symbiodinium types within the host habitat when conditions are chronically stressful. Previous studies have observed enhanced photosynthesis and fitness by clade C holobionts at non-stressful temperatures, relative to clade D. Yet, carbon-centered metrics cannot account for enhanced growth rates and patterns of symbiont succession to other genetic types when nitrogen often limits reef productivity. To investigate the metabolic costs of hosting thermally tolerant symbionts, we examined the assimilation and translocation of inorganic ¹⁵N and ¹³C in the coral Acropora tenuis experimentally infected with either clade C (sub-type C1) or D Symbiodinium at 28 and 30 °C. We show that at 28 °C, C1 holobionts acquired 22% more ¹⁵N than clade D. However, at 30 °C, C1 symbionts acquired equivalent nitrogen and 16% less carbon than D. We hypothesize that C1 competitively excludes clade D in hospite via enhanced nitrogen acquisition and thus dominates coral populations despite warming oceans.     

The Effect of Simulating Different Intermediate Host Snail Species on the Link between Water Temperature and Schistosomiasis Risk

Introduction

A number of studies have attempted to predict the effects of climate change on schistosomiasis risk. The importance of considering different species of intermediate host snails separately has never previously been explored.

Methods

An agent-based model of water temperature and Biomphalaria pfeifferi population dynamics and Schistosoma mansoni transmission was parameterised to two additional species of snail: B. glabrata and B. alexandrina.

Results

Simulated B. alexandrina populations had lower minimum and maximum temperatures for survival than B. pfeifferi populations (12.5–29.5°C vs. 14.0–31.5°C). B. glabrata populations survived over a smaller range of temperatures than either B. pfeifferi or B. alexandrina (17.0°C–29.5°C). Infection risk peaked at 16.5°C, 25.0°C and 19.0°C respectively when B. pfeifferi, B. glabrata and B. alexandrina were simulated. For all species, infection risk increased sharply once a minimum temperature was reached.

Conclusions

The results from all three species suggest that infection risk may increase dramatically with small increases in temperature in areas at or near the currents limits of schistosome transmission. The effect of small increases in temperature in areas where schistosomiasis is currently found will depend both on current temperatures and on the species of snail acting as intermediate host(s) in the area. In most areas where B. pfeifferi is the host, infection risk is likely to decrease. In cooler areas where B. glabrata is the host, infection risk may increase slightly. In cooler areas where B. alexandrina is the host, infection risk may more than double with only 2°C increase in temperature. Our results show that it is crucial to consider the species of intermediate host when attempting to predict the effects of climate change on schistosomiasis.     

Investigating the causes and consequences of symbiont shuffling in a multi-partner reef coral symbiosis under environmental change

Dynamic symbioses may critically mediate impacts of climate change on diverse organisms, with repercussions for ecosystem persistence in some cases. On coral reefs, increases in heat-tolerant symbionts after thermal bleaching can reduce coral susceptibility to future stress. However, the relevance of this adaptive response is equivocal owing to conflicting reports of symbiont stability and change. We help reconcile this conflict by showing that change in symbiont community composition (symbiont shuffling) in Orbicella faveolata depends on the disturbance severity and recovery environment. The proportion of heat-tolerant symbionts dramatically increased following severe experimental bleaching, especially in a warmer recovery environment, but tended to decrease if bleaching was less severe. These patterns can be explained by variation in symbiont performance in the changing microenvironments created by differentially bleached host tissues. Furthermore, higher proportions of heat-tolerant symbionts linearly increased bleaching resistance but reduced photochemical efficiency, suggesting that any change in community structure oppositely impacts performance and stress tolerance. Therefore, even minor symbiont shuffling can adaptively benefit corals, although fitness effects of resulting trade-offs are difficult to predict. This work helps elucidate causes and consequences of dynamism in symbiosis, which is critical to predicting responses of multi-partner symbioses such as O. faveolata to environmental change.     

Honeybees prefer warmer nectar and less viscous nectar,regardless of sugar concentration

The internal temperature of flowers may be higher than air temperature, and warmer nectar could offer energetic advantages for honeybee thermoregulation, as well as being easier to drink owing to its lower viscosity. We investigated the responses of Apis mellifera scutellata (10 colonies) to warmed 10% w/w sucrose solutions, maintained at 20–35°C, independent of low air temperatures, and to 20% w/w sucrose solutions with the viscosity increased by the addition of the inert polysaccharide Tylose (up to the equivalent of 34.5% sucrose). Honeybee crop loads increased with nectar temperature, as did the total consumption of sucrose solutions over 2 h by all bees visiting the feeders. In addition, the preference of marked honeybees shifted towards higher nectar temperatures with successive feeder visits. Crop loads were inversely proportional to the viscosity of the artificial nectar, as was the total consumption of sucrose solutions over 2 h. Marked honeybees avoided higher nectar viscosities with successive feeder visits. Bees thus showed strong preferences for both warmer and less viscous nectar, independent of changes in its sugar concentration. Bees may benefit from foraging on nectars that are warmer than air temperature for two reasons that are not mutually exclusive: reduced thermoregulatory costs and faster ingestion times due to the lower viscosity.     

Diversity and functional analysis of Chinese bumblebee gut microbiota reveal the metabolic niche and antibiotic resistance variation of Gilliamella

Bumblebees play an important role in maintaining the balance of natural and agricultural ecosystems,and the characteristic gut microbiota of bumblebees exhibit significant mutualistic functions.China has the highest diversity of bumblebees;however,gut microbiota of Chinese bumblebees have mostly been investigated through cultureindependent studies.Here,we analyzed the gut communities of bumblebees from Sichuan,Yunnan,and Shaanxi provinces in China through 16S ribosomal RNA amplicon sequencing and bacterial isolation.It revealed that the bumblebees examined in this study harbored two gut enterotypes as previously reported:one is dominated by Gilliamella and Snodgrassella,and the other is distinguished by prevalent environmental species.The gut compositions obviously varied among different individual bees.We then isolated 325 bacterial strains and the comparative genomic analysis of Gillianiella strains revealed that galactose and pectin digestion pathways were conserved in strains from bumblebees,while genes for the utilization of arabinose,mannose,xylose,and rhamnose were mostly lost.Only two strains from the Chinese bumblebees possess the multidrug-resistant gene emrB,which is phylogenetically closely related to that from the symbionts of soil entomopathogenic nematode.In contrast,tetracycline-resistant genes were uniquely present in three strains from the USA.Our results illustrate the prevalence of strain-level variations in the metabolic potentials and the distributions of antibiotic-resistant genes in Chinese bumblebee gut bacteria.     

Can Winter-Active Bumblebees Survive the Cold? Assessing the Cold Tolerance of Bombus terrestris audax and the Effects of Pollen Feeding

There is now considerable evidence that climate change is disrupting the phenology of key pollinator species. The recently reported UK winter activity of the bumblebee Bombus terrestris brings a novel set of thermal challenges to bumblebee workers that would typically only be exposed to summer conditions. Here we assess the ability of workers to survive acute and chronic cold stress (via lower lethal temperatures and lower lethal times at 0°C), the capacity for rapid cold hardening (RCH) and the influence of diet (pollen versus nectar consumption) on supercooling points (SCP). Comparisons are made with chronic cold stress indices and SCPs in queen bumblebees. Results showed worker bees were able to survive acute temperatures likely to be experienced in a mild winter, with queens significantly more tolerant to chronic cold temperature stress. The first evidence of RCH in any Hymenoptera is shown. In addition, dietary manipulation indicated the consumption of pollen significantly increased SCP temperature. These results are discussed in the light of winter active bumblebees and climate change.     

Flexible associations between Pocillopora corals and Symbiodinium limit utility of symbiosis ecology in defining species

Corals in the genus Pocillopora are the primary framework builders of eastern tropical Pacific (ETP) reefs. These corals typically associate with algal symbionts (genus Symbiodinium) in clade C and/or D, with clade D associations having greater thermal tolerance and resistance to bleaching. Recently, cryptic "species" delineations within both Pocillopora and Symbiodinium have been suggested, with host–symbiont specificity used as a supporting taxonomic character in both genera. In particular, it has been suggested that three lineages of Pocillopora (types 1–3) exist in the ETP, of which type 1 is the exclusive host of heat-tolerant Symbiodinium D1. This host specificity has been used to support the species name "Symbiodinium glynni" for this symbiont. To validate these host–symbiont relationships and their taxonomic utility, we identified Pocillopora types and their associated Symbiodinium at three sites in the ETP. We found greater flexibility in host–symbiont combinations than previously reported, with both Pocillopora types 1 and 3 able to host and be dominated by Symbiodinium in clade C or D. The prevalence of certain combinations did vary among sites, showing that a gradient of specificity exists which may be mediated by evolutionary relationships and environmental disturbance history. However, these results limit the utility of apparent host–symbiont specificity (which may have been a result of undersampling) in defining species boundaries in either corals or Symbiodinium. They also suggest that a greater diversity of corals may benefit from the thermal tolerance of clade D symbionts, affirming the need to conserve Pocillopora across its entire geographic and environmental range.     

Persistence of F-Specific RNA Coliphages in Surface Waters from a Produce Production Region along the Central Coast of California

F+ RNA coliphages (FRNA) are used to source-track fecal contamination and as surrogates for enteric pathogen persistence in the environment. However, the environmental persistence of FRNA is not clearly understood and necessitates the evaluation of the survival of prototype and environmental isolates of FRNA representing all four genogroups in surface waters from the central coast of California. Water temperature played a significant role in persistence–all prototype and environmental strains survived significantly longer at 10°C compared to 25°C. Similarly, the availability of host bacterium was found to be critical in FRNA survival. In the absence of E. coli F_amp, all prototypes of FRNA disappeared rapidly with a D-value (days for one log reduction) of <1.2 d from water samples incubated at 25°C; the longest surviving prototype was SP. However, in the presence of the host, the order of persistence at 25°C was QB>MS2>SP>GA and at 10°C it was QB = MS2>GA>SP. Significant differences in survival were observed between prototypes and environmental isolates of FRNA. While most environmental isolates disappeared rapidly at 25°C and in the absence of the host, members of genogroups GIII and GI persisted longer with the host compared to members of GII and GIV. Consequentially, FRNA based source tracking methods can be used to detect phages from recent fecal contamination along with those that persist longer in the environment as a result of cooler temperatures and increased host presence.     

Tropical Strains of Ralstonia solanacearum Outcompete Race 3 Biovar 2 Strains at Lowland Tropical Temperatures

Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands.     

Loss of symbiont infectivity following thermal stress can be a factor limiting recovery from bleaching in cnidarians

Increases in seawater temperature can cause coral bleaching through loss of symbiotic algae (dinoflagellates of the family Symbiodiniaceae). Corals can recover from bleaching by recruiting algae into host cells from the residual symbiont population or from the external environment. However, the high coral mortality that often follows mass-bleaching events suggests that recovery is often limited in the wild. Here, we examine the effect of pre-exposure to heat stress on the capacity of symbiotic algae to infect cnidarian hosts using the Aiptasia (sea-anemone)-Symbiodiniaceae model system. We found that the symbiont strain Breviolum sp. CS-164 (ITS2 type B1), both free-living and in symbiosis, loses the capacity to infect the host following exposure to heat stress. This loss of infectivity is reversible, however, a longer exposure to heat stress increases the time taken for reversal. Under the same experimental conditions, the loss of infectivity was not observed in another strain Breviolum psygmophilum CCMP2459 (ITS2 type B2). Our results suggest that recovery from bleaching can be limited by the loss of symbiont infectivity following exposure to heat stress.Subject terms: Microbial ecology, Biodiversity

Cnidarians including reef-building corals harbor endosymbiotic dinoflagellates of the family Symbiodiniaceae, from which they derive the majority of their energy. Therefore, the breakdown of the symbiotic relationship, a process known as bleaching, can result in the host starving. However, bleaching is not always lethal because symbiont densities can recover [1, 2]. Recovery from bleaching is driven mainly by symbiotic algae that remain within the bleached corals (the residual population) dividing and spreading throughout the colony [3], and also possibly through the recruitment of free-living symbiotic algae from the external environment [4]. In the last few decades, coral cover has drastically decreased in many regions, due to frequent mass coral bleaching events caused by global warming [5], implying that recovery from bleaching is often limited by unknown factors. In the present study, we demonstrate that both free-living and residual symbiont cells lose their capacity to infect cnidarian host cells once they are exposed to high temperature stress, and present this mechanism as a limiting factor for the host’s recovery from bleaching.We first examined the effect of pre-exposure to high temperature on infectivity using aposymbiotic Exaiptasia pallida (or “Aiptasia”) polyps (Supplementary Fig. 1) and cultured strains of Breviolum sp. CS-164 (ITS2 type B1). Symbiotic algae and polyps were separately incubated at either 25 or 32 °C for 3 days. Following this initial treatment, polyps were inoculated with symbiotic algae at 25 °C for 3 days. Infectivity was then determined by counting the number of algae in the tentacles where algal colonization occurs quickly and individual symbiont cells are easily visualized [6]. When both symbiotic algae and hosts were pre-exposed to 25 °C, significant numbers of algae were seen in tentacles (Fig. 1a). In contrast, the number (Fig. 1a) and density (Fig. 1b) of algae in the tentacles were significantly lower when both the algae and the host were exposed simultaneously to 32 °C, and when the algae alone were exposed to 32 °C. Thus, symbiotic algae, but not host polyps, lose their capacity to form a symbiotic relationship once they are exposed to high temperature. Neither cell viability measured by Evans blue staining (Supplementary Figs. 2a, b and 3) nor cell density (Supplementary Fig. 2c) differed between CS-164 cells exposed to 25 and 32 °C, indicating that infectivity was not lost by the lethal damage to cells. We repeated this experiment with another strain, B. psygmophilum CCMP2459 (ITS2 type B2). In contrast to the results with CS-164, high temperature had no effect on infectivity of CCMP2459 (Supplementary Fig. 4a). These results demonstrate that symbiotic algae can lose their capacity to infect host cells following exposure to high temperature and that thermal sensitivity differs between these two algal strains (Fig. 1b and Supplementary Fig. 4a).Open in a separate windowFig. 1Loss of infectivity in Breviolum sp. CS-164 following exposure to elevated temperature.a Fluorescent photographs of Aiptasia polyps 3 days after culturing with symbiont cells in four different treatments (i) neither symbionts nor polyps exposed to high temperature (32 °C) for 3 days, (ii) only symbionts exposed to high temperature, (iii) only polyps exposed to high temperature, (iv) both symbionts and polyps exposed to high temperature. Red dots show chlorophyll fluorescence from algal symbionts. b The density of symbionts in tentacles was measured 3 days after culturing Aiptasia polyps (H) with symbiont cells (S) in four different treatments, as indicated below the panel and outlined in the text. c The density of symbionts was measured 3 days after culturing Aiptasia polyps with symbiotic algae in different treatments. In this experiment, symbiotic algae that had been expelled from Aiptasia polyps cultured at 25 or 32 °C for 3 days were used to infect Aiptasia. b, c Values are log₂ fold changes with respect to the samples without any temperature treatment. Each point represents an independent experiment. ns, not significant (with p > 0.05); **, p < 0.01.We next examined whether the loss of infectivity following pre-exposure to high temperature also occurs when symbiotic algae are in symbiosis with the host rather than free living (Fig. 1c and Supplementary Fig. 4b). We prepared symbiotic Aiptasia polyps with either CCMP2459 or CS-164 by separately inoculating them in aposymbiotic polyps, and then exposed each group to either 25 or 32 °C for 3 days. Algae expelled from the polyps during this treatment were collected and then used to inoculate aposymbiotic Aiptasia at 25 °C. In CS-164, after 3 days of inoculation, symbiont density in Aiptasia became lower with algae collected at 32 °C than 25 °C (Fig. 1c). However, in CCMP2459, there was no difference in the infectivity between algae collected at 25 and 32 °C (Supplementary Fig. 4b). Our results demonstrate that symbiont cells, both free-living and in symbiosis, can lose infectivity following exposure to high temperature and that thermal sensitivity differs between these two algal strains.We then tested whether or not the temperature-induced loss of infectivity was reversible (Fig. 2). Free-living CS-164 cells were pre-exposed to 25 or 32 °C for either 2 or 3 days after which they were allowed to recover for a maximum of 10 days at 25 °C. After these treatments, symbiotic algae were used to inoculate aposymbiotic Aiptasia polyps at 25 °C for 3 days. Cells with pre-exposure to 32 °C for 2 days had lower infectivity but regained the capacity to infect host cells after a 5-day recovery period (Fig. 2). However, after 3 days exposure, infectivity gradually recovered but remained lower than the controls even after 10 days (Fig. 2). Our results demonstrate that the loss of infectivity following temperature stress is reversible in algal cells but a longer exposure to heat stress increases the time taken to reverse the loss of infectivity.Open in a separate windowFig. 2Reversibility of the lost infectivity upon the exposure to elevated temperature in Breviolum sp. CS-164.The density of symbionts in tentacles was measured following treatments as described in the text and shown by the relative to control. Values are log₂ fold changes with respect to the control. The box and line represent the quartiles and median, respectively. Each point represents an independent experiment. *, p < 0.05.In the present study, infectivity was tested by introducing symbiotic algae directly into the host’s body cavity, suggesting that the loss of infectivity seen in our experiments is likely due to a failure of the host to take up the algal cells via phagocytosis or for symbiont cells to persist within host cells. Factors such as symbiont cell size [7] and the symbiont surface glycome [8, 9] are potentially important determinants of symbiont uptake and persistence, though we still know little about this topic (see review [10]). Furthermore, it is unknown how these various discriminatory factors are influenced by thermal stress.In coral larvae and juveniles, the initiation of symbiosis with algae is reduced at high temperatures, suggesting that global warming will complicate the relationship between host and symbiont [11–13]. However, the mechanism for this reduction in the rate of symbiosis establishment is not clear. Our results suggest that the loss of symbiont infectivity is one possible cause of this phenomenon.Recovery of symbiont densities following coral bleaching relies on a supply of symbiont cells either from within the host or from the external environment. Our results show that symbiotic algae, both free-living and symbiotic, lose the capacity to infect the host following exposure to high temperature stress (Fig. 1). This loss of infectivity is reversible but dependent on the duration of the thermal stress (Fig. 2). Thus, following coral bleaching events, especially those induced by thermal anomalies that can last for weeks, symbiont densities within the host are unlikely to recover in time to avoid the host starving due to physiological compromise of the symbionts, rather than the host. Nonetheless, given the differences in sensitivity between two strains tested (Fig. 1b and Supplementary Fig. 4a), if heat tolerant symbionts are available in the environment, this might provide a chance for recovery.     

Cold-Active Chemoorganotrophic Bacteria from Permanently Ice-Covered Lake Hoare, McMurdo Dry Valleys, Antarctica

Eight strains of chemoorganotrophic bacteria were isolated from the water column of Lake Hoare, McMurdo Dry Valleys, Antarctica, using cold enrichment temperatures. The isolates were Alpha-, Beta-, and Gammaproteobacteria and Actinobacteria spp. All isolates grew at 0°C, and all but one grew at subzero temperatures characteristic of the water column of Lake Hoare. Growth temperature optima varied among isolates, but the majority showed optima near 15°C, indicative of cold-active phenotypes. One isolate was truly psychrophilic, growing optimally around 10°C and not above 20°C. Half of the isolates grew at 2% salt while the other half did not, and all but one isolate grew at 2 atm of O₂. Our isolates are the first prokaryotes from the water column of Lake Hoare to be characterized phylogenetically and physiologically and show that cold-active species of at least two major phyla of Bacteria inhabit Lake Hoare.