Okadaic acid: a potential defense molecule for the sponge <Emphasis Type="Italic">Suberites domuncula</Emphasis>

Sponges are sessile filter feeders that are exposed to toxic compounds/xenobiotics and a huge number of diverse pro- and eukaryotic organisms occurring in the aqueous milieu. To cope with these threats, sponges have developed a variety of chemical and immunological defense systems. Among them are potent secondary metabolites or toxins. The demosponge Suberites domuncula contains the bioactive compound okadaic acid; concentrations of ᅢ ng/g (ᅬ nM) of wet tissue have been quantified by LC/MS. In the present study, we sought to determine whether okadaic acid is beneficial for this sponge. Antibodies were raised against okadaic acid and used to localize this compound in situ. It could be demonstrated that the polyclonal antibodies/antiserum stained the sponge cells (endopinacocytes) which form the epithelium of the lacunae and the water channels. They also reacted with bacteria present in sponge tissue. Okadaic acid was found to augment the immune response of S. domuncula against the bacterial endotoxin lipopolysaccharide (LPS). If primmorphs, a special form of 3-D aggregates, are incubated with LPS at a concentration of 3 µg/ml, a reduction in the incorporation rate of [³H] phenylalanine is measured. In the presence of okadaic acid, this effect is measured already at a concentration of 1 µg/ml, suggesting that okadaic acid sensitizes this (immune) response. Okadaic acid is a known inhibitor of protein phosphatases which, as shown here, cause an increased phosphorylation of p38, a central kinase of the MAP kinase pathway; this may explain the sensitive response of the sponge cells to LPS. At higher concentrations, okadaic acid induces apoptosis, as was demonstrated here by a differential expression of the Bcl-2 homolog and the caspase gene. It is concluded that okadaic acid has two functions in the sponge; first, at low concentrations (<100 nM) a stimulation of the defense system against bacteria, and second, at concentrations above 500 nM, a differential effect on the pro-/anti-apoptotic genes.     

Chemical ecology and origin of defensive compounds in the Antarctic nudibranch Austrodoris kerguelenensis (Opisthobranchia: Gastropoda)

The common Antarctic nudibranch Austrodoris kerguelenensis (Bergh) contains diterpene diacylglycerides only present in its external body parts. These compounds provide a chemical defense against sympatric predators, such as the seastar Odontaster validus Koehler. Bioassays conducted with O. validus revealed that live nudibranchs, mantle tissue and Et₂O extract of the A. kerguelenensis mantle deterred feeding by the seastar. Further bioassays testing organic fractions of the Et₂O mantle extract showed that the diterpene diacylglycerides, as well as corresponding monoacylglycerides and monoacylglycerides of regular fatty acids, were responsible for the feeding deterrence in O. validus. We suggest that A. kerguelenensis derives the bioactive diacylglycerides by de novo biosynthesis rather than by sequestration from its sponge diet, since the mollusk does not contain active metabolites in the viscera, and neither the active compounds nor precursors were detected in the sponge diet. Furthermore, A. kerguelenensis did not show a strong chemodetection or feeding preference for its main diet, hexactinellid sponges, in Y-maze and food choice experiments, respectively.     

Host specificity of the symbiotic cyanobacterium<Emphasis Type="Italic"> Oscillatoria spongeliae</Emphasis> in marine sponges,<Emphasis Type="Italic"> Dysidea</Emphasis> spp.

Marine sponges can host a variety of cyanobacterial and bacterial symbionts, but it is often unclear whether these symbionts are generalists that occur in many host species or specialists that occur only in certain species or populations of sponges. The filamentous cyanobacterium Oscillatoria spongeliae is found in the sponges Dysidea n. sp. aff. herbacea 1A and 1B, and similar cyanobacteria are found in D. n. sp. aff. granulosa. We amplified and sequenced sponge nuclear ribosomal DNA (rDNA) and cyanobacterial 16S rDNA from specimens of these three sponges. We then used these sequences to construct phylogenies for host sponges and their symbiotic cyanobacteria. Each of these three sponge species hosts a distinct cyanobacterial clade, suggesting a high degree of host specificity and potential coevolution between symbiotic cyanobacteria and their host sponges.     

Consistent association of crenarchaeal Archaea with sponges of the genus Axinella

The association of sponges with microorganisms has been accepted to be a common feature of the phylum Porifera. Herein we describe the association between filamentous Archaea and three Mediterranean species of sponges from the family Axinellidae (Porifera: Demospongiae). Axinella damicornis, A. verrucosa and Axinella sp. harbor a high concentration of filamentous Archaea in the collagen that surrounds the siliceous spicules that form their skeleton. These Archaea have been found in every axinellid specimen studied, regardless of their environment and collection time. The morphology of the filaments has been studied using transmission electron microscopy, and they all show similar characteristics. Their nature has been determined by in situ hybridization experiments and by PCR amplification and sequencing of their 16S DNA. Each sponge species contains a single filamentous archaeal phylotype. The Archaea of the three sponges are closely related to each other and to the marine "group 1" crenarchaeotes. Our findings suggest that this newly described association could be defined as a symbiosis, where biochemical and/or metabolic relationships between the sponge hosts and their symbionts remain to be determined.     

The importance of micro and macro morphological variation in the adaptation of a sublittoral demosponge to current extremes

Sponges are known to show morphological acclimation in response to habitat variation. However, previous studies have concentrated on only one aspect of morphological variation, either gross morphology or spicule morphology. Cliona celata (Grant) is common in a variety of different habitats on the south-west coast of Ireland and has been investigated with respect to morphological variability on both scales. C. celata exhibited six different gross morphological body forms (ridged, burrowing, massive, massive/chimneys, encrusting, encrusting/chimneys). The body form exhibited was correlated to local environment, showing the extent of morphological adaptation in C. celata. Sponge size varied (from 548ᇟ to 2,345녹 cm²) between sites, with the largest (2,345녹 cm²) being found at the most stable site where flow rates were <5 cm^-1 (F>23.24, P<0.05). This may seem paradoxical as growth conditions were considered poor, but mortality and damage from material in suspension was reduced at low energy sites. At the spicule level, morphological variation was also present. Spicules at high energy sites were significantly longer, narrower and less numerous than at low energy sites (F>15.36, P<0.05). Previously, spicule variation has been associated with increased stiffness in hostile environments. However, longer, thinner spicules, as found in C. celata, may result in a more flexible sponge. This is the first study to show both gross morphological (macro) and spicule (micro) variation in a single species of sponge. However, this study only reinforces some of the previously produced information on both of these adaptations of sponges to varying environments. This study also illustrates how the results of single studies should not be used to draw conclusions for group level adaptation.     

Cryptic speciation in a high gene flow scenario in the oviparous marine sponge Chondrosia reniformis

Sponge systematics has been traditionally based on the study of the skeleton (spicules and spongin fibres). However, sponges of the genus Chondrosia are devoid of those skeletal features, making it difficult to distinguish between different species in the genus. Chondrosia reniformis Nardo, 1847, the type species of the genus, was described from the Mediterranean Sea. The lack of distinguishing morphological features may have been responsible for the widespread assignment of specimens of the genus to this species; as a result C. reniformis is considered to be a cosmopolitan species. In this work, populations of C. reniformis from the western Mediterranean (France) and the West Atlantic (Bermuda and Brazil) were analysed using allozyme electrophoresis for 13 enzyme loci. Levels of mean heterozygosity were high (Bermuda and Brazil H=0.27 and W Mediterranean H=0.12), as is often observed in sponge species. Gene identities observed between West Atlantic and Mediterranean populations were low (I=0.40-0.52, typical values for congeneric species), including the presence of four diagnostic loci. This level of divergence clearly shows that they are not conspecific. Hence, a worldwide or cosmopolitan distribution of C. reniformis would seem improbable. However, the West Atlantic samples (Bermuda and Brazil) were genetically similar (gene identity, I=0.88-0.95) over a distance of 8,000 km. This is the first report of genetic homogeneity in a sponge species over such a large geographical distance.     

Precopulatory assessment of male quality in an arctiid moth (Utetheisa ornatrix): hydroxydanaidal is the only criterion of choice

Females of the moth Utetheisa ornatrix (Lepidoptera: Arctiidae) mate preferentially with males that excel in three quantitatively correlated attributes: body mass, systemic content of defensive pyrrolizidine alkaloid (derived from the larval diet), and glandular content of the courtship pheromone hydroxydanaidal (derived from the alkaloid). By so choosing, the females obtain direct phenotypic benefits (alkaloid and nutrient received with the spermatophore), and indirect genetic benefits (genes for large size, a heritable trait). We asked whether the female appraises the courting male on the basis of all three attributes, or whether, as had been postulated, she does so on the basis of the intensity of the pheromonal scent alone. We present data indicating that male possession of hydroxydanaidal is indeed the sole criterion of choice. Females fail to differentiate between males that differ in body mass or alkaloid content if the males lack hydroxydanaidal, but choose between males that are size-matched and alkaloid-free if one of the males has been experimentally endowed with hydroxydanaidal. We show moreover that females are able to differentiate between males that contain unequal quantities of hydroxydanaidal. Females abide by these criteria whether or not they themselves contain alkaloid. Their choice was also unaffected by whether they were confined singly with 2 males in small mating chambers, or were in groups of 10 with 20 males in large flight cages.     

Induction of larval settlement and metamorphosis of the sea urchin Strongylocentrotus intermedius by glycoglycerolipids from the green alga Ulvella lens

In aquaculture centers of the northern region of Japan, "Nami-ita" (waved polycarbonate plates), on which the green alga Ulvella lens Crouan frat. (Chaetophoraceae: Chaetophorales) was cultured, are used to promote larval settlement and metamorphosis of the sea urchin species Strongylocentrotus intermedius (A. Agassiz) and S. nudus (A. Agassiz). We investigated chemical inducer(s) for larval settlement and metamorphosis of these sea urchins with extracts of U. lens. Bioassay-guided separation of the methanol extract using a combination of column and thin-layer chromatography led to the isolation of several active compounds, the chemical structures of which were determined by spectral and chemical methods. These active compounds were identified as glycoglycerolipids, all comprising several molecular species: sulfoquinovosyl monoacylglycerols (SQMGs), sulfoquinovosyl diacylglycerols (SQDGs), monogalactosyl monoacylglycerols (MGMGs), monogalactosyl diacylglycerols (MGDGs), digalactosyl monoacylglycerols (DGMGs) and digalactosyl diacylglycerols (DGDGs). Among these glycolipids, SQMGs, MGMGs, MGDGs and DGMGs induced larval metamorphosis of the sea urchin S. intermedius. SQMGs and MGDGs induced larval metamorphosis at a concentration of 5 µg ml^-1, whereas SQDGs and DGDGs only induced larval settlement. These glycoglycerolipids are new congeners of chemical inducers to settlement and metamorphosis of planktonic larvae of sea urchins. The findings would provide a better understanding of larval settlement and metamorphosis in sea urchins.     

Alarm responses of the green sea urchin, Strongylocentrotus droebachiensis, induced by chemically labelled durophagous predators and simulated acts of predation

The green sea urchin, Strongylocentrotus droebachiensis, exhibited immediate behavioural responses to waterborne chemosensory cues from two durophagous predators, the Atlantic wolffish, Anarhichas lupus, and the edible crab, Cancer pagurus; as well as from crushed conspecifics and crushed blue mussels, Mytilus edulis. The response patterns were dose dependent and diet dependent. Strong responses were elicited by water conditioned by echinivorous wolffish (97.5%), undiluted urchin extract (73%), undiluted mussel extract (52.5%), and by water conditioned by echinivorous crab (45%). In contrast, urchin extract diluted to 1%, mussel extract diluted to 10%, and water conditioned by predators on a mussel diet elicited weak responses (~20%). Infection by the endoparasitic nematode Echinomermella matsi had no significant effect on the response pattern of S. droebachiensis. There was no conclusive evidence of an alarm response to the predators per se, as the weak response to stimuli from non-echinivorous wolffish and crab, as well as from the seastar Asterias rubens, may have been caused by chemical cues transmitted from their mussel diet. The diet-dependent response to predators suggests that active predators were labelled by chemical cues from their echinoid prey. The chemical cue from echinivorous wolffish acted as both an arrestant and as a repellent, whereas the response to other cues was predominantly or exclusively repellent. The response to echinivorous wolffish was quantitatively stronger than, and qualitatively different from, the response to other stimuli, including undiluted urchin extract. The wolffish is apparently being labelled by a latent chemical cue which derives its potency from activation by, or interaction with, substances in the digestive system of the wolffish. We interpret this novel phenomenon as evidence of alarm signal magnification. The induced behavioural modifications demonstrate the green sea urchins' ability to detect chemical cues associated with active durophagous predators, and would therefore seem to have adaptive potential as a predator defence mechanism.     

Molecular response of the sponge Suberites domuncula to bacterial infection

The aim of this study was the documentation of the molecular immune response of Suberites domuncula upon bacterial infection. Additionally, the bacteria that are naturally present in the sponge after prolonged aquarium maintenance were characterized. After 6 months of maintenance of S. domuncula in seawater aquaria, only one bacterial 16S rDNA sequence could be recovered, which belongs to the genus Pseudomonas. Concomitantly, morphologically uniform bacteria were found encapsulated in bacteriocytes. These findings indicate that certain bacteria, possibly of the genus Pseudomonas, are able to persist for long periods in host bacteriocytes. Subsequent to performing a previously established infection assay with S. domuncula, a potentially pathogenic Vibrio sp. was isolated from the tissues. Furthermore, the host tissue disintegrated and asexual propagation bodies (gemmules) were formed. In order to gain insights into the molecular events occurring after bacterial infection, the stress-response kinases, p38 protein kinase and JNK protein kinase, were analyzed. It is demonstrated that these two kinases are activated (phosphorylated) upon incubation of the tissue with the bacterial endotoxin lipopolysaccharide (LPS). Moreover, LPS strongly inhibits protein synthesis. It is concluded that there are many functionally different interactions between S. domuncula and bacteria and that the animal possesses mechanisms to differentiate between bacteria and to respond accordingly.     

The 1998 bleaching event and its aftermath on a coral reef in Belize

Widespread thermal anomalies in 1997-1998, due primarily to regional effects of the El Niño-Southern Oscillation and possibly augmented by global warming, caused severe coral bleaching worldwide. Corals in all habitats along the Belizean barrier reef bleached as a result of elevated sea temperatures in the summer and fall of 1998, and in fore-reef habitats of the outer barrier reef and offshore platforms they showed signs of recovery in 1999. In contrast, coral populations on reefs in the central shelf lagoon died off catastrophically. Based on an analysis of reef cores, this was the first bleaching-induced mass coral mortality in the central lagoon in at least the last 3,000 years. Satellite data for the Channel Cay reef complex, the most intensively studied of the lagoonal reefs, revealed a prolonged period of elevated sea-surface temperatures (SSTs) in the late summer and early fall of 1998. From 18 September to 1 October 1998, anomalies around this reef averaged +2.2°C, peaking at 4.0°C above the local HotSpot threshold. In situ temperature records from a nearby site corroborated the observation that the late summer and early fall of 1998 were extraordinarily warm compared to other years. The lettuce coral, Agaricia tenuifolia, which was the dominant occupant of space on reef slopes in the central lagoon, was nearly eradicated at Channel Cay between October 1998 and January 1999. Although the loss of Ag. tenuifolia opened extensive areas of carbonate substrate for colonization, coral cover remained extremely low and coral recruitment was depressed through March 2001. High densities of the sea urchin Echinometra viridis kept the cover of fleshy and filamentous macroalgae to low levels, but the cover of an encrusting sponge, Chondrilla cf. nucula, increased. Further increases in sponge cover will impede the recovery of Ag. tenuifolia and other coral species by decreasing the availability of substrate for recruitment and growth. If coral populations are depressed on a long-term basis, the vertical accretion of skeletal carbonates at Channel Cay will slow or cease over the coming decades, a time during which global-warming scenarios predict accelerated sea-level rise.     

Stimulation of soil microbial activity by essential oils

Summary. Although essential oils are well known antimicrobial agents, some microorganisms are activated by them and can use them as a carbon and energy source; this is the case for soil bacteria from Mediterranean ecosystems. We examined the assumption that soil microorganisms when offered with an essential oil, to which they had been previously exposed, would respond faster making immediate use of the newly added substrate. Origanum vulgare subsp. hirtum, Rosmarinus officinalis, Mentha spicata, and Coridothymus capitatus plants were collected and their essential oils isolated. Soil samples from the upper surface layer, beneath these aromatic plants, were also collected. All possible combinations of essential oils and soil samples were examined as well as the effect of the oil of R. officinalis and the non-indigenous, Lavandula angustifolia, on soil samples collected from cultivated fields. Soil respiration was used as a measure of the microbial activity. Oils (0.1 ml) were repeatedly added to the soil samples (150 g) and CO₂ release was measured every seven days. Essential oils differed in their chemical composition. In spite of that, they activated respiration of the different soil samples, even of those not previously exposed to essential oils, to a comparable degree. These results suggest that essential oils are used as a carbon and energy source by rather ubiquitously occurring soil microorganisms and provide evidence that they would not accumulate in the soil, if environmental conditions favour growth of these microorganisms.     

Involvement of quinones and phenoloxidase in the allorejection reaction in a colonial ascidian, Botrylloides simodensis: histochemical and immunohistochemical study

When incompatible colonies of Botrylloides simodensis were brought into contact at their artificially cut surfaces, allorejection occurred and a black line was formed along the contact border. Morula cells (MCs), a type of hemocytes, are the major effector in the allorejection reaction and are known to possess phenoloxidase (PO) that generates quinones. In this rejection reaction, MCs infiltrate the tunic and break down, discharging their vacuolar contents. Ascorbic acid (antioxidant) and benzamidine (protease inhibitor) showed inhibitory effects on MC breakdown, black line formation and new tunic cuticle formation, whereas tropolone (metal chelator) and sodium benzoate (substrate analog) did not. MCs probably store some amount of quinones, as well as PO; oxidants derived from the quinones appear to disintegrate the tissue to form a black line and promote MC breakdown. Histochemical and immunohistochemical studies revealed that MCs contain eosinophilic materials, PO and quinones. Quinones that are stored in MCs and produced by PO probably have a destructive function, forming rejection lesions.     

Dynamics of pigment degradation by the copepodite stage of <Emphasis Type="Italic">Pseudodiaptomus</Emphasis><Emphasis Type="Italic">euryhalinus</Emphasis> feeding on <Emphasis Type="Italic">Tetraselmis</Emphasis><Emphasis Type="Italic">suecica</Emphasis>

Short-term (3 h) changes in concentration of chlorophylls and their derivatives in stage V Pseudodiaptomus euryhalinus and their fecal material were followed by HPLC during a 24 h experiment. Copepodites were fed with the prasinophyte Tetraselmis suecica. Intact chlorophyll a and b were found in animals and fecal material and had similar dynamics of accumulation over time. The extent of transformation of chlorophyll a and b to colorless compounds was different with chlorophyll a being more extensively degraded. Additionally, several chlorophyll derivatives (pheophytin and pyropheophytin-like pigments) were found. Pyropheophytin a was the most abundant followed by pheophytin b, pheophytin a, and pheophorbide a. Relative amounts of pheopigments were different in copepodites and fecal material, and pheophytin a, pheophorbide a, and pheophytin b concentrations were low and variable. The amount of ingested chlorophyll recovered as chlorophyll a and its derivatives in fecal and copepodite pools was generally low (<5%), with one exception occurring after 9 h, when it accounted for >70%. These data suggest individual pheopigments are produced at different rates and that chemical or enzymatic mechanisms in the gut of copepodites act on the two chlorophylls in different ways.     

Worker policing in the bee Apis florea

Apis florea is a single-combed, open-nesting, dwarf honeybee indigenous to Asia. In common with other species of this genus, A. florea is highly polyandrous, and is therefore predicted to curtail worker reproduction by mutual policing mechanisms that keep worker reproduction at an extremely low level. Policing mechanisms could involve destruction of workers' eggs or offspring, or aggression toward those workers that are reproductively active. We show that in A. florea, worker-laid eggs are eliminated approximately twice as fast as queen-laid eggs, indicating that A. florea uses oophagy of worker-laid eggs as a mechanism of worker policing. Genetic analysis of four colonies indicated that all males produced were sons of queens, not workers. Dissections of 800 workers, from four colonies, did not reveal any significant levels of ovary activation. These results suggest that worker policing is an effective component of the mechanisms that maintain worker sterility in this species. Furthermore, they suggest that worker policing via oophagy of worker-laid eggs is pleisiomorphic for the genus.     

Effects of chemical cues on orientation of blue crab, <Emphasis Type="Italic">Callinectes</Emphasis><Emphasis Type="Italic">sapidus</Emphasis>, megalopae in flow: implications for location of nursery areas

Megalopae (postlarvae) of the blue crab Callinectes sapidus Rathbun use flood-tide transport (FTT) for movement into and up estuaries. Since they settle around the time of slack water at the end of flood tide during FTT, it was predicted that orientation toward primary nursery areas of aquatic vegetation occurs at this time. This study tested the hypotheses that megalopae locate nursery areas by swimming upstream in the presence of chemical odors from potential nursery areas and avoid adverse microhabitats by swimming downstream when predator or adverse environmental odors are present. Megalopae were tested in a flume where they were exposed to the sequence of cues mediating FTT (i.e. 2 psu increase in salinity followed by an increase and a decrease in current speed and turbulence). The flume contained odor water either from the developmental area (offshore water), nursery area vegetation (seagrass, Zostera marina; salt marsh cord grass, Spartina alterniflora), predators (fiddler crab, Uca pugilator; mud crab, Panopeus herbstii; grass shrimp, Palaemonetes pugio), or chemicals associated with adverse environments (ammonium). Vertical positions of premolt and intermolt megalopae were similar in water devoid of estuarine chemical cues (offshore water) and water containing seagrass odor. Upstream swimming behavior (orientation) of intermolt megalopae was also similar in these waters. However, there was an ontogenetic behavioral change, as the proportion of premolt megalopae oriented upstream generally increased as the concentration of seagrass and salt marsh cord grass odor increased and as current speed decreased. Upstream orientation of premolt megalopae in response to seagrass odor decreased significantly (i.e. downstream swimming increased) in the presence of odor from U. pugilator, P. pugio, and ammonium, but not from P. herbstii. Thus, the hypothesis was supported. These results suggest premolt megalopae orient toward nursery areas by swimming upstream in response to odors from aquatic vegetation as current speeds decrease at the end of nocturnal flood tides. Moreover, these results also indicate that megalopae may discriminate among microhabitats and avoid adverse settlement habitat, as orientation toward nursery areas is reversed by predator odors and ammonium.     

Cuticular hydrocarbons and reproductive status in the social wasp Polistes dominulus

Most species of social insect are characterized by a reproductive division of labor among morphologically specialized individuals. In contrast, there exist many species where all individuals are morphologically identical and dominance relationships determine which individuals mate and/or reproduce. In newly founded multiple-foundress associations of the social wasp Polistes dominulus, foundresses establish dominance hierarchies where the top-ranked (alpha) female monopolizes egg laying. The possibility that chemical cues are used for recognition of egg-laying individuals has not been explored in this wasp. Using non-destructive techniques, we examined the relationship between ovarian activity and the proportions of cuticular hydrocarbons of three female types (dominant and subordinate foundresses and workers) in 11 colonies. Immediately after nest foundation, no differences were found between alpha and subordinate females. In contrast, at worker emergence, alpha females were statistically distinguishable from both subordinates and workers. We experimentally removed the alpha female in 5 of the original nests and reanalyzed hydrocarbon proportions of the new dominant individual. Replacement individuals were all found to acquire a cuticular signature characteristic of the alpha female. This suggests that cuticular hydrocarbons are used as cues of ovarian activity in P. dominulus, and we discuss our results in terms of a switch from behavioral dominance to chemical signaling in this wasp.     

Possible involvement of the glycocalyx in the ichthyotoxicity of Chattonella marina (Raphidophyceae): immunological approach using antiserum against cell surface structures of the flagellate

Although the ichthyotoxic mechanism of Chattonella marina is still unknown, several lines of evidence suggest that the reactive oxygen species (ROS), such as superoxide anion (O₂^-), hydrogen peroxide (H₂O₂)_, and hydroxyl radical (·OH), produced by C. marina are involved in the mortality of fish exposed to this flagellate. Recently, we found that the cell-free supernatant prepared from C. marina, which is considered to contain the glycocalyx, showed NADPH-dependent O₂^- generation. In this study, we prepared antiserum against the crude glycocalyx of C. marina. Using indirect immunofluorescence, it was confirmed that the antiserum specifically reacted with C. marina cells. In addition to C. marina, the antiserum also reacted with other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica, whereas no reactivity was observed against six other flagellate species tested. These results suggest that raphidophycean flagellates have common epitopes recognized by the antiserum. Interestingly, immunohistochemical analysis of paraformaldehyde-fixed gill lamellae from yellowtail exposed to C. marina revealed that the antiserum stained the surface of gill lamellae, while no such staining pattern was observed in control gill lamellae. These results suggest that the glycocalyx may be discharged when C. marina cells are inhaled into the fishes' mouths and then come into contact with the gill surface. Based on the present results, together with our previous findings, we propose that continuous accumulation of the discharged glycocalyx on the gill surface occurs during C. marina exposure, which may be responsible for the ROS-mediated severe gill tissue damage leading to fish death.     

Responses of the flea beetles Phyllotreta nemorum and P. cruciferae to metabolically engineered Arabidopsis thaliana with an altered glucosinolate profile

Summary. Insects feeding on Cruciferae recognize their host plants at least partially by means of specific responses to glucosinolates. However, the effects of variations in glucosinolate levels on the acceptability of plants for specialized insects are not well understood. A survey of the literature demonstrated positive, no, as well as negative correlations between plant acceptability and glucosinolate levels. The present study took advantage of the presence of transgenic Arabidopsis thaliana plants with increased glucosinolate levels. Transgenic A. thaliana contain the CYP79A1 gene from Sorghum bicolor. This gene encodes an enzyme which converts L-tyrosine into p-hydroxyphenylacetaldoxime in the biosynthesis of cyanogenic glycosides in S. bicolor. In transgenic A. thaliana plants, endogenous enzymes convert p-hydroxyphenylacetaldoxime into p-hydroxybenzylglucosinolate (sinalbin), which is not found naturally in this plant. The introduction of CYP79A1 resulted in a four-fold increase in total glucosinolate levels in transgenic A. thaliana plants. Although these changes in glucosinolate levels were rather dramatic, they did not have any effects on the acceptability of A. thaliana for the two flea beetle species, Phyllotreta nemorum and P. cruciferae. The flea beetles did not discriminate between transgenic and wildtype plants. Furthermore, they did not discriminate between leaf discs of wildtype plants where different concentrations of p-hydroxybenzylglucosinolate had been applied topically on the leaf surface. Feeding in P. nemorum was stimulated by extremely high levels of allylglucosinolate while this compound had no effect on P. cruciferae. It is concluded that the effect of glucosinolates on adapted insects depends on the chemical or physical environment in which the glucosinolates are found.