Aposymbiotic Larvae Research Articles

Extracellular symbiont colonizes insect during embryo development.

Insects typically acquire their beneficial microbes early in development. Endosymbionts housed intracellularly are commonly integrated during oogenesis or embryogenesis, whereas extracellular microbes are only known to be acquired after hatching by immature instars such as larvae or nymphs. Here, however, we report on an extracellular symbiont that colonizes its host during embryo development. Tortoise beetles (Chrysomelidae: Cassidinae) host their digestive bacterial symbiont Stammera extracellularly within foregut symbiotic organs and in ovary-associated glands to ensure its vertical transmission. We outline the initial stages of symbiont colonization and observe that although the foregut symbiotic organs develop 3 days prior to larval emergence, they remain empty until the final 24h of embryo development. Infection by Stammera occurs during that timeframe and prior to hatching. By experimentally manipulating symbiont availability to embryos in the egg, we describe a 12-h developmental window governing colonization by Stammera. Symbiotic organs form normally in aposymbiotic larvae, demonstrating that these Stammera-bearing structures develop autonomously. In adults, the foregut symbiotic organs are already colonized following metamorphosis and host a stable Stammera population to facilitate folivory. The ovary-associated glands, however, initially lack Stammera. Symbiont abundance subsequently increases within these transmission organs, thereby ensuring sufficient titers at the onset of oviposition ~29days following metamorphosis. Collectively, our findings reveal that Stammera colonization precedes larval emergence, where its proliferation is eventually decoupled in adult beetles to match the nutritional and reproductive requirements of its host.

Performance of Orbicella faveolata larval cohorts does not align with previously observed thermal tolerance of adult source populations.

Orbicella faveolata, commonly known as the mountainous star coral, is a dominant reef-building species in the Caribbean, but populations have suffered sharp declines since the 1980s due to repeated bleaching and disease-driven mortality. Prior research has shown that inshore adult O. faveolata populations in the Florida Keys are able to maintain high coral cover and recover from bleaching faster than their offshore counterparts. However, whether this origin-specific variation in thermal resistance is heritable remains unclear. To address this knowledge gap, we produced purebred and hybrid larval crosses from O. faveolata gametes collected at two distinct reefs in the Upper Florida Keys, a nearshore site (Cheeca Rocks, CR) and an offshore site (Horseshoe Reef, HR), in two different years (2019, 2021). We then subjected these aposymbiotic larvae to severe (36°C) and moderate (32°C) heat challenges to quantify their thermal tolerance. Contrary to our expectation based on patterns of adult thermal tolerance, HR purebred larvae survived better and exhibited gene expression profiles that were less driven by stress response under elevated temperature compared to purebred CR and hybrid larvae. One potential explanation could be the compromised reproductive output of CR adult colonies due to repeated summer bleaching events in 2018 and 2019, as gametes originating from CR in 2019 contained less storage lipids than those from HR. These findings provide an important counter-example to the current selective breeding paradigm, that more tolerant parents will yield more tolerant offspring, and highlight the importance of adopting a holistic approach when evaluating larval quality for conservation and restoration purposes.

Enhancing Coral Settlement Through a Novel Larval Feeding Protocol

Intensifying anthropogenic stressors have contributed to declines in reef-building corals in many regions. These disturbances result in reduced live coral cover, impacting key population-level processes such as coral larval settlement and recruitment that are essential for reef recovery. Reef restoration efforts that rely on enhanced larval supply provide a pathway for the recovery of degraded reefs. However, corals at very early life stages experience high post-settlement mortality bottlenecks, which impede stock-recruitment processes. Overcoming these bottlenecks is a high priority goal in coral restoration. Some coral larvae are known to be capable of gaining exogenous nutrients. Therefore, we hypothesised that the capacity to access exogenous nutrients may confer advantages to larval survival, settlement and post-settlement success. The present study aimed to quantify the effect of larval feeding on coral larvae settlement and early post-settlement survival. We completed an ex-situ experiment using aposymbiotic larvae of two broadcast spawning reef-building coral species - Acropora tenuis and Acropora millepora. Larvae were randomly assigned to either fed or unfed treatment groups for each species. Fed larvae received homogenised Artemia once a day, for three days. Results show that for both species, feeding significantly increased larval settlement. Feeding A. millepora larvae more than doubled mean settlement (13.0 ± 1.17 SE vs 31.4 ± 2.88 SE; p <0.001). Similarly, feeding A. tenuis larvae increased mean settlement from 18.2 ( ± 1.85 SE) to 29.9 ( ± 2.22 SE; p <0.001). Larval feeding had an immediate positive effect on spat survival, such that A. millepora and A. tenuis spat from fed treatments had increased survival three days post-settlement (89.5% ± 3.75 SE vs 70.6% ± 2.59 SE, p <0.001; 88.8% ± 2.21 SE vs 71.4% ± 3.80 SE, p <0.001, respectively). Therefore, enhancing settlement and early post-settlement survival by feeding larvae homogenised Artemia has the potential to improve the effectiveness of larval rearing protocols and coral restoration efforts.

The early acquisition of symbiotic algae benefits larval survival and juvenile growth in the coral Acropora tenuis.

Larval dispersal and postsettlement survival of corals play significant roles in the maintenance of coral populations. Most corals acquire their symbiotic algae (Symbiodiniaceae) from the environment in each generation (horizontal transmission). For horizontal transmitters, the quick establishment of symbiosis is important for their survival, since the photosynthetic activity of symbiotic algae provides energy. However, recent studies have indicated that oxidative stress resulting from photosynthesis might also harm coral larvae. Therefore, it remains unclear whether symbionts contribute energy sources along with intrinsic lipids from eggs and assist in settlement/metamorphosis in early life stages. In the present study, we show that symbiotic algae contribute supplemental energy and are also associated with settlement. Furthermore, although juveniles acquired symbiotic algae after settlement, the acquisition of symbiotic algae in the larval stages caused higher growth (number of polyps and size) and low mortality in the juvenile stage. Our data suggest that symbiotic larvae potentially have longer dispersal periods due to their lower lipid consumption rates, which make them better able to retain buoyancy and motility, increasing the ability of symbiotic larvae to settle in favored locations compared with aposymbiotic larvae. Moreover, postsettlement juveniles may continue to benefit from symbiotic relationships formed during the larval stage. Overall, these findings reveal that the effects of symbiotic algae on Acropora tenuis coral larvae are beneficial, particularly under normal seawater temperature conditions.

Hypoxia as a physiological cue and pathological stress for coral larvae.

Ocean deoxygenation events are intensifying worldwide and can rapidly drive adult corals into a state of metabolic crisis and bleaching-induced mortality, but whether coral larvae are subject to similar stress remains untested. We experimentally exposed apo-symbiotic coral larvae of Acropora selago to deoxygenation stress with subsequent reoxygenation aligned to their night-day light cycle, and followed their gene expression using RNA-Seq. After 12h of deoxygenation stress (~2mg O2 /L), coral planulae demonstrated a low expression of HIF-targeted hypoxia response genes concomitant with a significantly high expression of PHD2 (a promoter of HIFα proteasomal degradation), similar to corresponding adult corals. Despite exhibiting a consistent swimming phenotype compared to control samples, the differential gene expression observed in planulae exposed to deoxygenation-reoxygenation suggests a disruption of pathways involved in developmental regulation, mitochondrial activity, lipid metabolism, and O2 -sensitive epigenetic regulators. Importantly, we found that treated larvae exhibited a disruption in the expression of conserved HIF-targeted developmental regulators, for example, Homeobox (HOX) genes, corroborating how changes in external oxygen levels can affect animal development. We discuss how the observed deoxygenation responses may be indicative of a possible acclimation response or alternatively may imply negative latent impacts for coral larval fitness.

Transmission of Bacterial Symbionts With and Without Genome Erosion Between a Beetle Host and the Plant Environment.

Many phytophagous insects harbor symbiotic bacteria that can be transmitted vertically from parents to offspring, or acquired horizontally from unrelated hosts or the environment. In the latter case, plants are a potential route for symbiont transfer and can thus foster a tripartite interaction between microbe, insect, and plant. Here, we focus on two bacterial symbionts of the darkling beetle Lagria villosa that belong to the genus Burkholderia; the culturable strain B. gladioli Lv-StA and the reduced-genome strain Burkholderia Lv-StB. The strains can be transmitted vertically and confer protection to the beetle’s eggs, but Lv-StA can also proliferate in plants, and both symbiont strains have presumably evolved from plant pathogens. Notably, little is known about the role of the environment for the transmission dynamics and the maintenance of the symbionts. Through manipulative assays, we demonstrate the transfer of the symbionts from the beetle to wheat, rice and soybean plants, as well as leaf litter. In addition, we confirm that aposymbiotic larvae can pick up Lv-StA from dry leaves and the symbiont can successfully establish in the beetle’s symbiotic organs. Also, we show that the presence of plants and soil in the environment improves symbiont maintenance. These results indicate that the symbionts of L. villosa beetles are still capable of interacting with plants despite signatures of genome erosion and suggest that a mixed-mode of bacterial transmission is likely key for the persistence of the symbiosis.

Can Acropora tenuis larvae attract native Symbiodiniaceae cells by green fluorescence at the initial establishment of symbiosis?

Most corals acquire symbiodiniacean symbionts from the surrounding environment to initiate symbiosis. The cell densities of Symbiodiniaceae in the environment are usually low, and mechanisms may exist by which new coral generations attract suitable endosymbionts. Phototaxis of suitable symbiodiniacean cells toward green fluorescence in corals has been proposed as one such mechanism. In the present study, we observed the phototaxis action wavelength of various strains of Symbiodiniaceae and the fluorescence spectra of aposymbiotic Acropora tenuis larvae at the time of endosymbiont uptake. The phototaxis patterns varied among the Symbiodiniaceae species and "native" endosymbionts-commonly found in Acropora juveniles present in natural environments; that is, Symbiodinium microadriaticum was attracted to blue light rather than to green light. Another native endosymbiont, Durusdinium trenchii, showed no phototaxis specific to any wavelength. Although the larvae exhibited green and broad orange fluorescence under blue-violet excitation light, the maximum green fluorescence peak did not coincide with that of the phototaxis action spectrum of S. microadriaticum. Rather, around the peak wavelength of larval green fluorescence, this native endosymbiont showed slightly negative phototaxis, suggesting that the green fluorescence of A. tenuis larvae may not play a role in the initial attraction of native endosymbionts. Conversely, broad blue larval fluorescence under UV-A excitation covered the maximum phototaxis action wavelength of S. microadriaticum. We also conducted infection tests using native endosymbionts and aposymbiotic larvae under red LED light that does not excite visible larval fluorescence. Almost all larvae failed to acquire S. microadriaticum cells, whereas D. trenchii cells were acquired by larvae even under red illumination. Thus, attraction mechanisms other than visible fluorescence might exist, at least in the case of D. trenchii. Our results suggest that further investigation and discussion, not limited to green fluorescence, would be required to elucidate the initial attraction mechanisms.

Coxiella-Like Endosymbiont of Rhipicephalus sanguineus Is Required for Physiological Processes During Ontogeny.

Obligatory hematophagous arthropods such as lice, bugs, flies, and ticks harbor bacterial endosymbionts that are expected to complement missing essential nutrients in their diet. Genomic and some experimental evidence support this expectation. Hard ticks (Acari: Ixodidae) are associated with several lineages of bacterial symbionts, and very few were experimentally shown to be essential to some aspects of tick’s fitness. In order to pinpoint the nature of interactions between hard ticks and their symbionts, we tested the effect of massive elimination of Coxiella-like endosymbionts (CLE) by antibiotics on the development and fitness of the brown dog tick (Rhipicephalus sanguineus). Administration of ofloxacin to engorged (blood fed) nymphs resulted in significant and acute reduction of their CLE loads – an effect that also persisted in subsequent life stages (aposymbiotic ticks). As a result, the post-feeding development of aposymbiotic female (but not male) nymphs was delayed. Additionally, aposymbiotic adult females needed a significantly prolonged feeding period in order to replete (detach from host), and had reduced engorgement weight and a lower capacity to produce eggs. Consequently, their fecundity and fertility were significantly reduced. Eggs produced by aposymbiotic females were free of CLE, and the resulting aposymbiotic larvae were unable to feed successfully. Our findings demonstrate that the observed fitness effects are due to CLE reduction and not due to antibiotic administration. Additionally, we suggest that the contribution of CLE is not mandatory for oocyte development and embryogenesis, but is required during feeding in females, when blood meal processing and tissue buildup are taking place. Presumably, under these extreme physiological demands, CLE contribute to R. sanguineus through supplementing essential micro- and macronutrients. Further nutrient complementary studies are required to support this hypothesis.

Increased apical sodium-dependent glucose transporter abundance in the ctenidium of the giant clam Tridacna squamosa upon illumination.

Giant clams contain phototrophic zooxanthellae, and live in nutrient-deficient tropical waters where light is available. We obtained the complete cDNA coding sequence of a homolog of mammalian sodium/glucose cotransporter 1 (SGLT1) - SGLT1-like - from the ctenidium of the fluted giant clam, Tridacna squamosaSGLT1-like had a host origin and was expressed predominantly in the ctenidium. Molecular characterizations reveal that SGLT1-like of T. squamosa could transport urea, in addition to glucose, as other SGLT1s do. It has an apical localization in the epithelium of ctenidial filaments and water channels, and the apical anti-SGLT1-like immunofluorescence was stronger in individuals exposed to light than to darkness. Furthermore, the protein abundance of SGLT1-like increased significantly in the ctenidium of individuals exposed to light for 12 h, although the SGLT1-like transcript level remained unchanged. As expected, T. squamosa could perform light-enhanced glucose absorption, which was impeded by exogenous urea. These results denote the close relationships between light-enhanced glucose absorption and light-enhanced SGLT1-like expression in the ctenidium of T. squamosa Although glucose absorption could be trivial compared with the donation of photosynthates from zooxanthellae in symbiotic adults, SGLT1-like might be essential for the survival of aposymbiotic larvae, leading to its retention in the symbiotic stage. A priori, glucose uptake through SGLT1-like might be augmented by the surface microbiome through nutrient cycling, and the absorbed glucose could partially fulfill the metabolic needs of the ctenidial cells. Additionally, SGLT1-like could partake in urea absorption, as T. squamosa is known to conduct light-enhanced urea uptake to benefit the nitrogen-deficient zooxanthellae.

Protection of Coral Larvae from Thermally Induced Oxidative Stress by Redox Nanoparticles.

Coral reefs are one of the most biologically diverse and economically important ecosystems on earth. However, the destruction of coral reefs has been reported worldwide owing to rising seawater temperature associated with global warming. In this study, we investigated the potential of a redox nanoparticle (RNPO) to scavenge reactive oxygen species (ROS), which are overproduced under heat stress and play a crucial role in causing coral mortality. When reef-building coral (Acropora tenuis) larvae, without algal symbionts, were exposed to thermal stress at 33°C, RNPO treatment significantly increased the survival rate. Proteome analysis of coral larvae was performed using nano-liquid chromatography-tandem mass spectrometry for the first time. The results revealed that several proteins related to ROS-induced oxidative stress were specifically identified in A. tenuis larvae without RNPO treatment, whereas these proteins were absent in RNPO-treated larvae, which suggested that RNPO effectively scavenged ROS from A. tenuis larvae. Results from this study indicate that RNPO treatment can reduce ROS in aposymbiotic coral larvae and would be a promising approach for protecting corals from thermal stress.

Eggs and larvae of Acropora palmata and larvae of Porites astreoides contain high amounts of dimethylsulfoniopropionate

Coral holobionts, including their symbionts, are known to produce large amounts of dimethylsulfoniopropionate (DMSP), a compound which some corals use to mitigate oxidative stress; however, very little work has been done on the presence and use of DMSP in early life history stages of coral, such as in eggs and larvae. This study shows that eggs and larvae, from Acropora palmata, and brooded larvae, from Porites astreoides, also contain high amounts of DMSP. Eggs and larvae of wild A. palmata were collected and contained extremely high levels of DMSP: 1.2μmol per larva and 359μmol per 100μL eggs. Larvae of P. astreoides were collected in flow-through laboratory tanks, from wild-collected parent colonies, over the course of the larval release cycle. In brooded larvae of P. astreoides, the amount of DMSP in larvae ranged from 11.6 to 1510nmol per larva; larval DMSP peaked by the second night of release and then decreased over the following release nights. These high levels in aposymbiotic eggs and larvae of A. palmata and the peaking trend in symbiotic larvae of P. astreoides are suggestive of parental provisioning. Given the large amounts of DMSP found in eggs and larvae, this study provides further evidence that even early life stages of the coral holobiont may benefit from the presence of DMSP.

Endosymbionts escape dead hydrothermal vent tubeworms to enrich the free-living population

Theory predicts that horizontal acquisition of symbionts by plants and animals must be coupled to release and limited dispersal of symbionts for intergenerational persistence of mutualisms. For deep-sea hydrothermal vent tubeworms (Vestimentifera, Siboglinidae), it has been demonstrated that a few symbiotic bacteria infect aposymbiotic host larvae and grow in a newly formed organ, the trophosome. However, whether viable symbionts can be released to augment environmental populations has been doubtful, because (i) the adult worms lack obvious openings and (ii) the vast majority of symbionts has been regarded as terminally differentiated. Here we show experimentally that symbionts rapidly escape their hosts upon death and recruit to surfaces where they proliferate. Estimating symbiont release from our experiments taken together with well-known tubeworm density ranges, we suggest a few million to 1.5 billion symbionts seeding the environment upon death of a tubeworm clump. In situ observations show that such clumps have rapid turnover, suggesting that release of large numbers of symbionts may ensure effective dispersal to new sites followed by active larval colonization. Moreover, release of symbionts might enable adaptations that evolve within host individuals to spread within host populations and possibly to new environments.

Early uptake of specific symbionts enhances the post-settlement survival of Acropora corals

For corals that establish symbioses with dinoflagellate Symbiodinium spp. at the lar- val stage or later through horizontal transmission, the ecological significance of the early uptake of algal symbionts remains unknown. It has been hypothesized that early uptake of symbionts is an advantage for long-distance dispersal. Here, we tested the hypothesis that early acquisition of symbionts enhances post-settlement survival. We used a cultured strain of clade A Symbiodinium that was isolated from wild Acropora spat as the algal symbiont. Symbiotic and aposymbiotic Acropora larvae were prepared in the laboratory and settled on experimental plates in the field. The survival of settlers was monitored for 15 mo. Our results showed that more larval-stage set- tlers harbouring symbionts survived than those without, even when there was no difference in the initial density of settled larvae. We analysed the Symbiodinium clades harboured by the corals at 1 mo after settlement, and found that clade A was less abundant in the corals that grew from aposymbiotic larvae than in those that developed from symbiotic larvae. There was also a marked difference in coral survival between aposymbiotic and symbiotic larvae over this period. The higher survival rate of 'early uptake' corals was more pronounced on shaded plates. These results suggest that the early uptake of specific symbionts enhances post-settlement survival in dark places such as reef crevices, which are sites commonly settled by coral larvae.

Symbiosis initiation in the bacterially luminous sea urchin cardinalfish Siphamia versicolor

To determine how each new generation of the sea urchin cardinalfish Siphamia versicolor acquires the symbiotic luminous bacterium Photobacterium mandapamensis, and when in its development the S. versicolor initiates the symbiosis, procedures were established for rearing S. versicolor larvae in an aposymbiotic state. Under the conditions provided, larvae survived and developed for 28 days after their release from the mouths of males. Notochord flexion began at 8 days post release (dpr). By 28 dpr, squamation was evident and the caudal complex was complete. The light organ remained free of bacteria but increased in size and complexity during development of the larvae. Thus, aposymbiotic larvae of the fish can survive and develop for extended periods, major components of the luminescence system develop in the absence of the bacteria and the bacteria are not acquired directly from a parent, via the egg or during mouth brooding. Presentation of the symbiotic bacteria to aposymbiotic larvae at 8-10 dpr, but not earlier, led to initiation of the symbiosis. Upon colonization of the light organ, the bacterial population increased rapidly and cells forming the light-organ chambers exhibited a differentiated appearance. Therefore, the light organ apparently first becomes receptive to colonization after 1 week post-release development, the symbiosis is initiated by bacteria acquired from the environment and bacterial colonization induces morphological changes in the nascent light organ. The abilities to culture larvae of S. versicolor for extended periods and to initiate the symbiosis in aposymbiotic larvae are key steps in establishing the experimental tractability of this highly specific vertebrate and microbe mutualism.

From Parent to Gamete: Vertical Transmission of Symbiodinium (Dinophyceae) ITS2 Sequence Assemblages in the Reef Building Coral Montipora capitata

Parental effects are ubiquitous in nature and in many organisms play a particularly critical role in the transfer of symbionts across generations; however, their influence and relative importance in the marine environment has rarely been considered. Coral reefs are biologically diverse and productive marine ecosystems, whose success is framed by symbiosis between reef-building corals and unicellular dinoflagellates in the genus Symbiodinium. Many corals produce aposymbiotic larvae that are infected by Symbiodinium from the environment (horizontal transmission), which allows for the acquisition of new endosymbionts (different from their parents) each generation. In the remaining species, Symbiodinium are transmitted directly from parent to offspring via eggs (vertical transmission), a mechanism that perpetuates the relationship between some or all of the Symbiodinium diversity found in the parent through multiple generations. Here we examine vertical transmission in the Hawaiian coral Montipora capitata by comparing the Symbiodinium ITS2 sequence assemblages in parent colonies and the eggs they produce. Parental effects on sequence assemblages in eggs are explored in the context of the coral genotype, colony morphology, and the environment of parent colonies. Our results indicate that ITS2 sequence assemblages in eggs are generally similar to their parents, and patterns in parental assemblages are different, and reflect environmental conditions, but not colony morphology or coral genotype. We conclude that eggs released by parent colonies during mass spawning events are seeded with different ITS2 sequence assemblages, which encompass phylogenetic variability that may have profound implications for the development, settlement and survival of coral offspring.

Host gene response to endosymbiont and pathogen in the cereal weevil Sitophilus oryzae

BackgroundInsects thriving on nutritionally poor habitats have integrated mutualistic intracellular symbiotic bacteria (endosymbionts) in a bacteria-bearing tissue (the bacteriome) that isolates the endosymbionts and protects them against a host systemic immune response. Whilst the metabolic and physiological features of long-term insect associations have been investigated in detail over the past decades, cellular and immune regulations that determine the host response to endosymbionts and pathogens have attracted interest more recently.ResultsTo investigate bacteriome cellular specificities and weevil immune responses to bacteria, we have constructed and sequenced 7 cDNA libraries from Sitophilus oryzae whole larvae and bacteriomes. Bioinformatic analysis of 26,886 ESTs led to the generation of 8,941 weevil unigenes. Based on in silico analysis and on the examination of genes involved in the cellular pathways of potential interest to intracellular symbiosis (i.e. cell growth and apoptosis, autophagy, immunity), we have selected and analyzed 29 genes using qRT-PCR, taking into consideration bacteriome specificity and symbiosis impact on the host response to pathogens. We show that the bacteriome tissue accumulates transcripts from genes involved in cellular development and survival, such as the apoptotic inhibitors iap2 and iap3, and endosomal fusion and trafficking, such as Rab7, Hrs, and SNARE. As regards our investigation into immunity, we first strengthen the bacteriome immunomodulation previously reported in S. zeamais. We show that the sarcotoxin, the c-type lysozyme, and the wpgrp2 genes are downregulated in the S. oryzae bacteriome, when compared to aposymbiotic insects and insects challenged with E. coli. Secondly, transcript level comparison between symbiotic and aposymbiotic larvae provides evidence that the immune systemic response to pathogens is decreased in symbiotic insects, as shown by the relatively high expression of wpgrp2, wpgrp3, coleoptericin-B, diptericin, and sarcotoxin genes in aposymbiotic insects.ConclusionsLibrary sequencing significantly increased the number of unigenes, allowing for improved functional and genetic investigations in the cereal weevil S. oryzae. Transcriptomic analyses support selective and local immune gene expression in the bacteriome tissue and uncover cellular pathways that are of potential interest to bacteriocyte survival and homeostasis. Bacterial challenge experiments have revealed that the systemic immune response would be less induced in a symbiotic insect, thus highlighting new perspectives on host immunity in long-term invertebrate co-evolutionary associations.

Profiling gene expression responses of coral larvae (Acropora millepora) to elevated temperature and settlement inducers using a novel RNA-Seq procedure

Elevated temperatures resulting from climate change pose a clear threat to reef-building corals; however, the traits that might influence corals' survival and dispersal during climate change remain poorly understood. Global gene expression profiling is a powerful hypothesis-forming tool that can help elucidate these traits. Here, we applied a novel RNA-Seq protocol to study molecular responses to heat and settlement inducers in aposymbiotic larvae of the reef-building coral Acropora millepora. This analysis of a single full-sibling family revealed contrasting responses between short- (4-h) and long-term (5-day) exposures to elevated temperatures. Heat shock proteins were up-regulated only in the short-term treatment, while the long-term treatment induced the down-regulation of ribosomal proteins and up-regulation of genes associated with ion transport and metabolism (Ca(2+) and CO(3)(2-)). We also profiled responses to settlement cues using a natural cue (crustose coralline algae, CCA) and a synthetic neuropeptide (GLW-amide). Both cues resulted in metamorphosis, accompanied by differential expression of genes with known developmental roles. Some genes were regulated only by the natural cue, which may correspond to the recruitment-associated behaviour and morphology changes that precede metamorphosis under CCA treatment, but are bypassed under GLW-amide treatment. Validation of these expression profiles using qPCR confirmed the quantitative accuracy of our RNA-Seq approach. Importantly, qPCR analysis of different larval families revealed extensive variation in these responses depending on genetic background, including qualitative differences (i.e. up-regulation in one family and down-regulation in another). Future studies of gene expression in corals will have to address this genetic variation, which could have important adaptive consequences for corals during global climate change.

Early molecular responses of coral larvae to hyperthermal stress

Most of the work on the impact of elevated temperature and light on Symbiodinium-invertebrate symbioses have focused primarily on how the photosynthetic (algal) partner is impacted. Understanding how the same stresses affect the invertebrate host, however, is in its infancy. In this study, we re-examined the direct effect of elevated temperatures on the invertebrate host exploring the early transcriptional response of aposymbiotic (without algal symbionts) coral larvae. The temperatures tested in the experimental design were 24 degrees C (ambient seawater temperature), 28 degrees C and 31 degrees C; and the sampling points were 3 and 10 h after temperature exposure. We explored relative changes in transcription using a cDNA microarray constructed for the scleractinian coral, Acropora millepora, and containing 18 142 expressed sequence tag (EST) clones/8386 unigenes. Our study identified 29 genes that were significantly up- and down-regulated when A. millepora coral larvae were exposed to elevated temperatures. Down-regulation of several key components of DNA/RNA metabolism was detected implying inhibition of general cellular processes. The down-regulation of protein synthesis, however, was not simple and random, which suggested that the stress response was a more complicated adjustment of cellular metabolism. We identified four significant outcomes during the very early hours of the transcriptional response to hyperthermal stress in coral larvae. First, the expression of heat-shock proteins increased rapidly (within 3 h) in response to hyperthermal stress. Second, a fluorescent protein homologue, DsRed-type FP, decreased its expression in response to elevated temperature reinforcing a potential role as a molecular marker for monitoring hyperthermal stress in nature. Third, the down-regulation of a coral mannose-binding C-type lectin under elevated temperature suggests that heat stress might compromise some components of the coral immune defence and therefore might bring about susceptibility to pathogenic diseases. And last, genes involved in protecting cells against oxidative stress showed little response at the early hours to heat stress, supporting the proposal that up-regulation of cnidarian host oxidative stress genes may require reactive oxygen species generated by stressed algal symbionts.

Onset of symbiosis and distribution patterns of symbiotic dinoflagellates in the larvae of scleractinian corals

The establishment of symbiosis in early developmental stages is important for reef-building corals because of the need for photosynthetically derived nutrition. Corals spawn eggs and sperm, or brood planula larvae and shed them into the water. Some coral eggs or planulae directly inherit symbiotic dinoflagellates (Symbiodinium spp.) from their parents, while others acquire them at each generation. In most species examined to date, the larvae without dinoflagellates (aposymbiotic larvae) can acquire symbionts during the larval stage, but little is known regarding the timing and detailed process of the onset of symbiosis. We examined larval uptake of symbiotic dinoflagellates in nine species of scleractinian corals, the onset of symbiosis through the early larval stages, and the distribution pattern of symbionts within the larval host, while living and with histology, of two acroporid corals under laboratory conditions. The larvae acquired symbiotic dinoflagellates during the planktonic phase in all corals examined which included Acropora digitifera, A. florida, A. intermedia, A. tenuis, Isopora palifera, Favia pallida, F. lizardensis, Pseudosiderastrea tayamai, and Ctenactis echinata. The larvae of A. digitifera and A. tenuis first acquired symbionts 6 and 5 days after fertilization, respectively. In A. digitifera larvae, this coincided with the formation of an oral pore and coelenteron. The number of symbiotic dinoflagellates increased over the experimental periods in both species. To test the hypothesis that nutrients promotes symbiotic uptake, the number of incorporated dinoflagellates was compared in the presence and absence of homogenized Artemia sp. A likelihood ratio test assuming a log-linear model indicated that Artemia sp. had a significantly positive effect on symbiont acquisition. These results suggest that the acquisition of symbiotic dinoflagellates during larval stages is in common with many coral species, and that the development of both a mouth and coelenteron play important roles in symbiont acquisition.

Identification of the Weevil immune genes and their expression in the bacteriome tissue

BackgroundPersistent infections with mutualistic intracellular bacteria (endosymbionts) are well represented in insects and are considered to be a driving force in evolution. However, while pathogenic relationships have been well studied over the last decades very little is known about the recognition of the endosymbionts by the host immune system and the mechanism that limits their infection to the bacteria-bearing host tissue (the bacteriome).ResultsTo study bacteriome immune specificity, we first identified immune-relevant genes of the weevil Sitophilus zeamais by using suppressive subtractive hybridization (SSH) and then analyzed their full-length coding sequences obtained by RACE-PCR experiments. We then measured immune gene expression in the bacteriome, and in the aposymbiotic larvae following S. zeamais primary endosymbiont (SZPE) injection into the hemolymph, in order to consider the questions of bacteriome immune specificity and the insect humoral response to symbionts. We show that larval challenge with the endosymbiont results in a significant induction of antibacterial peptide genes, providing evidence that, outside the bacteriome, SZPE are recognized as microbial intruders by the host. In the bacteriome, gene expression analysis shows the overexpression of one antibacterial peptide from the coleoptericin family and, intriguingly, homologs to genes described as immune modulators (that is, PGRP-LB, Tollip) were also shown to be highly expressed in the bacteriome.ConclusionThe current data provide the first description of immune gene expression in the insect bacteriome. Compared with the insect humoral response to SZPE, the bacteriome expresses few genes among those investigated in this work. This local immune gene expression may help to maintain the endosymbiont in the bacteriome and prevent its invasion into insect tissues. Further investigations of the coleoptericin, the PGRP and the Tollip genes should elucidate the role of the host immune system in the maintenance and regulation of endosymbiosis.