Physiological and Biochemical Performances of Menthol-Induced Aposymbiotic Corals

The unique mutualism between corals and their photosynthetic zooxanthellae (Symbiodinium spp.) is the driving force behind functional assemblages of coral reefs. However, the respective roles of hosts and Symbiodinium in this endosymbiotic association, particularly in response to environmental challenges (e.g., high sea surface temperatures), remain unsettled. One of the key obstacles is to produce and maintain aposymbiotic coral hosts for experimental purposes. In this study, a simple and gentle protocol to generate aposymbiotic coral hosts (Isopora palifera and Stylophora pistillata) was developed using repeated incubation in menthol/artificial seawater (ASW) medium under light and in ASW in darkness, which depleted more than 99% of Symbiodinium from the host within 4∼8 days. As indicated by the respiration rate, energy metabolism (by malate dehydrogenase activity), and nitrogen metabolism (by glutamate dehydrogenase activity and profiles of free amino acids), the physiological and biochemical performances of the menthol-induced aposymbiotic corals were comparable to their symbiotic counterparts without nutrient supplementation (e.g., for Stylophora) or with a nutrient supplement containing glycerol, vitamins, and a host mimic of free amino acid mixture (e.g., for Isopora). Differences in biochemical responses to menthol-induced bleaching between Stylophora and Isopora were attributed to the former digesting Symbiodinium rather than expelling the algae live as found in the latter species. Our studies showed that menthol could successfully bleach corals and provided aposymbiotic corals for further exploration of coral-alga symbioses.     

The etiological agent of cytoplasmic incompatibility in Culex pipiens

All individuals of Culex pipiens (wide sense) which have been examined were infected with Wolbachia pipientis. Larvae reared in tetracycline were freed of these symbiotes and remained free (aposymbiotic) in future generations. When males were freed of their symbiotes, they no longer displayed incompatibility. Aposymbiotic males were compatible with all females, whether infected or not. Aposymbiotic females, on the other hand, laid fertile eggs after mating with aposymbiotic males, but not after mating with normal males. Most eggs laid by aposymbiotic females after mating with normal males showed no development at all, even though the females had been inseminated.     

Toxicological effects of biocides on symbiotic and aposymbiotic juveniles of the hermatypic coral Acropora tenuis

Reef-building (or hermatypic) corals harbor the symbiotic dinoflagellates Symbiodinium spp. (Alveolata, Dinophyceae, Gymnodiniales), and contribute to the accretion of coral reefs in tropical and sub-tropical zones. In this study, toxicological effects of three commonly used biocides (dichlorvos (DDVP), a commonly used insecticide; diuron (DCMU), a herbicide; and tributyltin chloride (TBT-Cl), an anti-fouling agent) on the hermatypic coral Acropora tenuis (Anthozoa, Hexacorallia, Scleractinia) were studied using juveniles in both aposymbiotic (symbiont-free) and symbiotic conditions. After exposure to the chemicals, abnormalities such as detachment of soft tissues from the skeleton and/or death were observed, as well as lowered uptake of symbiotic algae (in aposymbiotic juveniles) or reduction of the symbiont population in tentacles (in symbiotic juveniles). Significant reduction of the symbiont population in tentacles of symbiotic juveniles exposed to DDVP, DCMU and TBT-Cl was observed at the concentrations of 100, 10, and 1 μg/l, respectively. Results of this study suggested that symbiotic juveniles of A. tenuis are more sensitive than in the aposymbiotic condition to DDVP and DCMU, but not to TBT-Cl.     

Comparison of sarcosine and methionine sulfoxide levels in symbiotic and aposymbiotic larvae of two sibling species,Sitophilus oryzae L. and S. zeamais mots. (Coleoptera: Curculionidae)

Sarcosine and methionine sulfoxide were investigated in several wild or laboratory-reared symbiotic and aposymbiotic strains of Sitophilus oryzae and S. zeamais. The amino acid composition of fourth-instar larvae indicated that a high level in sarcosine found together with a low level of methionine sulfoxide were biochemical characteristics of the aposymbiotic state in this genus. Nutritional experiments demonstrated that the synthesis of these two amino acids depended on dietary precursors. Since sarcosine and methionine sulfoxide are both methionine derivatives, it is therefore suggested that methionine metabolism in Sitophilus larvae might differ according to the presence or the absence of the symbiotic bacteria.     

Roles of the endosymbiont and leishmanolysin-like molecules expressed by Crithidia deanei in the interaction with mammalian fibroblasts

Crithidia deanei is an insect trypanosomatid that harbors a bacterial endosymbiont in its cytoplasm. In this work, we have demonstrated the influence of the endosymbiont on the interaction of C. deanei with mammalian fibroblasts, also implicating the surface leishmanolysin-like molecules of C. deanei in this process. The wild strain of C. deanei expressed a higher amount (2-fold) of leishmanolysin-like molecules in the parasite surface than the aposymbiotic strain. The treatment of parasites with anti-leishmanolysin antibodies or the fibroblasts with purified leishmanolysin-like molecules from C. deanei significantly reduced the association index. The aposymbiotic strain of C. deanei presented interaction rates about 2- and 3-fold lower with fibroblasts than the endosymbiont-bearing counterpart after 1 and 2 h, respectively. However, the association indexes were similar after 3 and 4 h of interaction. Additionally, we observed a 2-fold increase in the association index after 24-96 h of parasite-fibroblast interaction when compared to the interaction process performed for 4 h, irrespective to the presence of the endosymbiont, suggesting that fibroblasts support multiplication and survival of C. deanei. Both parasite strains were able to induce fibroblast lysis. Interestingly, the wild strain led to a 2-fold increase in fibroblasts death in comparison to the aposymbiotic strain after 48-96 h. We also showed that both wild and aposymbiotic biotinylated live parasites recognized the same receptor in the fibroblast cells.     

Comparative composition of free,peptide and protein amino acids in symbiotic and aposymbiotic Sitophilus oryzae (coleoptera,curculionidae)

Free, peptide-bound and total amino acids were determined in symbiotic and aposymbiotic Sitophilus oryzae. The concentrations of free and peptide amino acids were higher, that of protein amino acids lower in aposymbiotic larvae (+30, +66 and −35%, respectively). In the free fraction, tyrosine concentration was the only one to be lower in aposymbiotic larvae (−41%) and could be a limiting factor for protein synthesis.In nymphs and adults, the compositions in symbiotic and aposymbiotic insects were very similar. A deficiency of tyrosine occurred however in aposymbiotic nymphs and of aspartate, glutamate, arginine, lysine and tyrosine in aposymbiotic adults. On the other hand, the composition in ovaries was unchanged.It is suggested that symbiotes could favour the synthesis of proteins in the larvae by increasing the efficiency of the conversion of phenylalanine in tyrosine.     

Copper accumulation and oxidative stress in the sea anemone, Aiptasia pallida, after waterborne copper exposure

Copper is a common marine pollutant yet its effects on symbiotic cnidarians are largely understudied. To further understand the impact of elevated copper concentrations on marine symbiotic organisms, toxicity tests were conducted using the model sea anemone, Aiptasia pallida, with and without its zooxanthellae symbiont. Symbiotic and aposymbiotic A. pallida were exposed to sublethal copper concentrations (0, 5, 15, and 50 µg/L) for 7 d and copper accumulation, behavior, and the activity of the oxidative stress enzymes, superoxide dismutase (SOD), and catalase (CAT) were measured. Additionally, acute 96-h toxicity tests were conducted to determine LC₅₀ values of the organisms after copper exposure. Both symbiotic and aposymbiotic A. pallida rapidly accumulated copper in a time and dose dependent manner. However, higher copper concentrations accumulated in the aposymbiotic as compared to the symbiotic A. pallida. In response to the highest two copper exposures (15 and 50 µg/L) symbiotic A. pallida upregulated CAT activity to combat the damaging effects of hydrogen peroxide. Contrary to these results, SOD activity significantly decreased during the highest copper exposure, when compared to controls. CAT activity was not detected and SOD was substantially (> 10 fold) reduced in aposymbiotic A. pallida, suggesting that the zooxanthellae are associated with the oxidative stress response. Copper exposure as low as 5 µg/L caused tentacle retraction and increased mucus production in both symbiotic and aposymbiotic anemones. The LC₅₀ values for symbiotic and aposymbiotic A. pallida exposed to copper for 96 h were 148 µg/L (95% confidence interval = 126.4, 173.8) and 206 µg/L (95% confidence interval = 175.2, 242.2), respectively. Understanding the varying responses of symbiotic and aposymbiotic A. pallida to copper stress may advance our comprehension of the functional roles of zooxanthellae and host. Although the mechanism of copper toxicity has not been fully elucidated, it is clear that A. pallida accumulate copper and are sensitive, as effects were detected at environmentally relevant copper concentrations. Likewise, A. pallida may be useful in biomonitoring copper polluted environments.     

Body size and symbiotic status influence gonad development in <Emphasis Type="Italic">Aiptasia pallida</Emphasis> anemones

Pale anemones (Aiptasia pallida) coexist with dinoflagellates (primarily Symbiodinium minutum) in a mutualistic relationship. The purpose of this study was to investigate the role of these symbionts in gonad development of anemone hosts. Symbiotic and aposymbiotic anemones were subjected to light cycles that induced gametogenesis. These anemones were then sampled weekly for nine weeks, and gonad development was analyzed histologically. Anemone size was measured as mean body column diameter, and oocytes or sperm follicles were counted for each anemone. Generalized linear models were used to evaluate the influence of body size and symbiotic status on whether gonads were present and on the number of oocytes or sperm follicles produced. Body size predicted whether gonads were present, with larger anemones being more likely than smaller anemones to develop gonads. Both body size and symbiotic status predicted gonad size, such that larger and symbiotic anemones produced more oocytes and sperm follicles than smaller and aposymbiotic anemones. Overall, only 22 % of aposymbiotic females produced oocytes, whereas 63 % of symbiotic females produced oocytes. Similarly, 6 % of aposymbiotic males produced sperm follicles, whereas 60 % of symbiotic males produced sperm follicles. Thus, while gonads were present in 62 % of symbiotic anemones, they were present in only 11 % of aposymbiotic anemones. These results indicate that dinoflagellate symbionts influence gonad development and thus sexual maturation in both female and male Aiptasia pallida anemones. This finding substantiates and expands our current understanding of the importance of symbionts in the development and physiology of cnidarian hosts.     

Viability of Culex pipiens pipiens eggs affected by nutrition and aposymbiosis

Raisins were a better source of carbohydrate than sucrose for reproduction by autogenous Culex pipiens. A blood meal increased the number of eggs per raft from 49 autogenously to 114. Eggs of aposymbiotic females produced autogenously did not hatch, but 34% of the eggs were viable if the mosquitoes fed on chickens. With repeated blood meals the number of eggs per raft and the rate of embryonation and hatching declined in each successive gonotrophic cycle. In about $\text{1}\text{4}$ of the unhatched eggs of normal females there were no fully developed embryos, while many more of the unhatched eggs of aposymbiotic females contained no evidence of embryonic development. After the fifth blood meal, neither normal nor aposymbiotic insects oviposited. The ovaries of the nulliparous females contained approximately 10% of the potential number of mature terminal oocytes. Proximally in the ovarioles there were dilatations and coiled tracheoles indicating egg resorption. There were fewer parous follicles in aposymbiotic than in normal females. Larval rearing water, i.e., infusion in which larvae had been reared, was more attractive than fresh infusion for oviposition by normal, autogenous mosquitoes. The degree of embryonation of the eggs was lower and the hatching success rate was poorer in fresh infusion than in larval rearing water.     

Vitamin B6 Generated by Obligate Symbionts Is Critical for Maintaining Proline Homeostasis and Fecundity in Tsetse Flies

The viviparous tsetse fly utilizes proline as a hemolymph-borne energy source. In tsetse, biosynthesis of proline from alanine involves the enzyme alanine-glyoxylate aminotransferase (AGAT), which requires pyridoxal phosphate (vitamin B₆) as a cofactor. This vitamin can be synthesized by tsetse''s obligate symbiont, Wigglesworthia glossinidia. In this study, we examined the role of Wigglesworthia-produced vitamin B₆ for maintenance of proline homeostasis, specifically during the energetically expensive lactation period of the tsetse''s reproductive cycle. We found that expression of agat, as well as genes involved in vitamin B₆ metabolism in both host and symbiont, increases in lactating flies. Removal of symbionts via antibiotic treatment of flies (aposymbiotic) led to hypoprolinemia, reduced levels of vitamin B₆ in lactating females, and decreased fecundity. Proline homeostasis and fecundity recovered partially when aposymbiotic tsetse were fed a diet supplemented with either yeast or Wigglesworthia extracts. RNA interference-mediated knockdown of agat in wild-type flies reduced hemolymph proline levels to that of aposymbiotic females. Aposymbiotic flies treated with agat short interfering RNA (siRNA) remained hypoprolinemic even upon dietary supplementation with microbial extracts or B vitamins. Flies infected with parasitic African trypanosomes display lower hemolymph proline levels, suggesting that the reduced fecundity observed in parasitized flies could result from parasite interference with proline homeostasis. This interference could be manifested by competition between tsetse and trypanosomes for vitamins, proline, or other factors involved in their synthesis. Collectively, these results indicate that the presence of Wigglesworthia in tsetse is critical for the maintenance of proline homeostasis through vitamin B₆ production.     

Development responses of symbiotic and aposymbiotic weevils Sitophilus oryzae L. (Coleoptera,curculionidae) to a diet supplemented with aromatic amino acids

Developmental times of symbiotic and aposymbiotic strains of the rice weevil Sitophilus oryzae were compared, in response to changes in concentration of phenylalanine or tyrosine in whole wheat flour pellets. Aposymbiotic insects were shown to require more aromatic amino acids than symbiotic insects, since a very low supply (0.1%) resulted in faster growth (11%). Incorporation results of [³H]-tyrosine during the larval and pupal stages indicated that total tyrosine intake was lower in aposymbiotic insects, but the incorporation into the cuticle of both strains did not significantly differ. It is suggested that the slower growth rate of weevils without symbiotes is due, in part, to a less efficient utilization of exogenous tyrosine (in the food) and to a lack of endogenous tyrosine (supplied by the symbiotes).     

Wolbachia sp. (Rickettsiales: Rickettsiaceae) a symbiont of the almond moth,Ephestia cautella: Ultrastructure and influence on host fertility

Wolbachia sp. is a maternally inherited symbiont of the almond moth, Ephestia cautella. It is transmitted through the cytoplasm of the egg and occurs normally in the gonads of all stages of the moth. The symbiont is responsible for reproductive cytoplasmic incompatibility between crosses of experimental laboratory strains of aposymbiotic female moths and symbiotic (normal) males. Although female moths were inseminated in laboratory tests, their eggs failed to hatch and exhibited no signs of embryonic development. The reciprocal cross, i.e., symbiotic female months × aposymbiotic males, produced normal progeny.The ultrastructure of Wolbachia was studied in sections of E. cautella larval testes. Symbionts, minute rod-shaped structures, were abundant in the cytoplasm of hypertrophied spermatids. There was no indication of deleterious influence of symbionts on sperm production or activity. Strains of Wolbachia occur in allopatric populations of insects where they may function as a genetic isolation mechanism. Microorganismal reproductive incompatibility has been suggested as a possible approach for insect control.     

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

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

Nudibranch-sponge feeding dynamics: Benefits of symbiont-containing sponge to Archidoris montereyensis (Cooper, 1862) and recovery of nudibranch feeding scars by Halichondria panicea (Pallas, 1766)

Selected interactions between the encrusting sponge Halichondria panicea and its primary predator, the dorid nudibranch Archidoris montereyensis, were investigated in a high-latitude rocky intertidal community spatially dominated by H. panicea. Feeding experiments were conducted in which A. montereyensis pairs were provided with sponge containing symbiotic zoochlorellae or sponge in which the zoochlorellae population had been reduced or removed by shading. Nudibranchs consuming H. panicea with symbiotic zoochlorellae had higher feeding, growth, and egg production rates than individuals eating aposymbiotic sponge. We simulated A. montereyensis predation on H. panicea by creating typically sized feeding grooves in the sponge. H. panicea's response was high linear growth rates into the experimental feeding grooves, generally recovering most of the groove area within 4 weeks. Overall, the sponge's rapid response to tissue damage minimizes grazing impacts and substrate loss and reduces susceptibility to wave removal.     

Nutrient availability for zooxanthellae derived from physiological activities of Condylactus spp

Differences in phosphate metabolism of symbiotic and aposymbiotic Condylactus suggest that the host animal makes available quantities of phosphate to support growth of zooxanthellae. Nitrite may serve as a nitrogen source for symbionts as indicated by host removal of nitrite from sea water.The presence of zooxanthellae is responsible for removal of phosphate from sea water in the dark whereas there is excretion during light periods. There is a greater uptake of nitrite from sea water in the light compared with the dark in symbiotic animals.Since nitrate is removed from sea water by aposymbiotic animals, the presence of nitrate reducing bacteria is proposed.     

Natural infections of aposymbiotic Cassiopea xamachana scyphistomae from environmental pools of Symbiodinium

The ability to acquire different types of the symbiotic dinoflagellate Symbiodinium (zooxanthellae) from the environment was investigated using aposymbiotic scyphistomae of the jellyfish Cassiopea xamachana. Non-symbiotic scyphistomae were placed on an offshore Florida patch reef and in Florida Bay during 3- and 5-day periods in March, and 5-day exposures in May, August and December of 2003. Scyphistomae were maintained in culture for several months, after which members of clades A, B, C and D Symbiodinium were detected in these hosts by denaturing gradient gel electrophoresis (DGGE) analyses. These findings contrast with naturally collected C. xamachana medusa from Florida Bay where all specimens possessed only Symbiodinium type A1. Furthermore, the polyps did not acquire the symbionts found in nearby cnidarian colonies, suggesting that a diverse pool of symbiont lineages exists in the environment. These results support previous laboratory studies where aposymbiotic hosts were initially non-selective and capable of acquiring many kinds of Symbiodinium. The specificity seen in adult hosts is likely a result of post-infection processes due to competitive exclusion or other mechanisms. A higher percentage of polyps became infected after 5 days of exposure, compared to 3 days, and no infections were observed in laboratory controls held in filtered seawater. Infections were lowest (50% at both sites) in March of 2003, when seawater temperatures were at their annual minima. Infection was 100% in scyphistomae exposed for 5 days during the months of May, August and December of 2003. These findings suggest that this host system, in addition to addressing questions of host-symbiont selectivity, can be employed to monitor and define the abundance and distribution of natural pools of Symbiodinium.     

Trypanosomatid protozoa: survey of acetylornithinase and ornithine acetyltransferase

Trypanosomatid protozoa (Crithidia deanei, C. deanei aposymbiotic, C. oncopelti, C. fasciculata, C. acanthocephali, Leptomonas seymouri, L. collosoma, L. samueli, Herpetomonas samuelpessoai, H. sp., H. megaseliae, H. muscarum muscarum, Leishmania donovani, L. braziliensis, Trypanosoma cruzi, T. conorhini and T. mega) were examined for the presence of acetylornithinase (EC 3.5.1.16) and ornithine acetyltransferase (EC 2.3.1.35). As a rule, species of the genus Crithidia presented one of the two enzymes for the conversion of acetylornithine into ornithine. Crithidia fasciculata and C. acanthocephali presented acetylornithinase, while C. deanei and C. oncopelti, species harboring symbionts, presented ornithine acetyltransferase. The enzyme was absent in the aposymbiotic strain of C. deanei, which suggests that the enzyme belongs to the symbiont. Among the other trypanosomatids examined only Herpetomonas samuelpessoai presented acetylomithinase. The participation of acetylornithinase and ornithine acetyltransferase in the metabolism of trypanosomatids is discussed in the light of their nutritional requirements and possession of enzymes of the arginineornithine metabolism.     

Detection of the Free-Living Forms of Sulfide-Oxidizing Gill Endosymbionts in the Lucinid Habitat (Thalassia testudinum Environment)

Target DNA from the uncultivable Codakia orbicularis endosymbiont was PCR amplified from sea-grass sediment. To confirm that such amplifications originated from intact bacterial cells rather than free DNA, whole-cell hybridization (fluorescence in situ hybridization technique) with the specific probe Symco2 was performed along with experimental infection of aposymbiotic juveniles placed in contact with the same sediment. Taken together, the data demonstrate that the sulfide-oxidizing gill endosymbiont of Codakia orbicularis is present in the environment as a free-living uncultivable form.     

Is dimethylsulfoniopropionate (DMSP) produced by the symbionts or the host in an anemone–zooxanthella symbiosis?

Many groups of tropical cnidarians including scleractinian corals, octocorals, corallimorphs, and anemones contain the tertiary sulfonium compound dimethylsulfoniopropionate (DMSP). It is not known if the compound is synthesized by the animals, their microalgal symbionts, or derived through their diet. We determined the source of the DMSP in several species of tropical and temperate anemones using three approaches: (1) conducting comparative measurements of DMSP in aposymbiotic and zooxanthellate anemones of three species that harbor zooxanthellae, and similar measurements in one species that can harbor both zooxanthellae and zoochlorellae, (2) manipulating the presence or absence of zooxanthellae by inoculating juvenile aposymbiotic anemones (Aiptasia pallida) with their symbiont, Symbiodinium bermudense, and (3) manipulating the numbers of S. bermudense by growing aposymbiotic and zooxanthellate A. pallida in the light and the dark. DMSP was present in zooxanthellate anemones in concentrations of 3.4–15 μmol g⁻¹ fresh mass (FM). In aposymbiotic Aiptasia spp. and Anthopleura elegantissima that lacked large numbers of zooxanthellae, concentrations ranged from being undetectable to 0.43 μmol g⁻¹ FM. When aposymbiotic A. pallida were inoculated with zooxanthellae, concentrations of DMSP were an average of 4.24 μmol g⁻¹ FM after 5 weeks; DMSP was undetectable in uninoculated control animals. Aposymbiotic anemones maintained in the light or the dark for 6 weeks contained no DMSP or zooxanthellae. Zooxanthellate anemones in the light contained five times as many zooxanthellae and approximately 7.5 times as much DMSP as zooxanthellate anemones maintained in the dark. Taken together, these data show that the zooxanthellae are the sole source of DMSP in A. pallida. The trends in DMSP concentrations in other species of zooxanthellate anemones suggest that this phenomenon is not limited to A. pallida but may be more generally true for other anemones or even other cnidarians hosting species of Symbiodinium. Communicated by Biology Editor Dr. Michael Lesser