Induction of metamorphosis in the Asian shore crab Hemigrapsus sanguineus: Characterization of the cue associated with biofilm from adult habitat

Recruitment of crabs to nursery habitat requires settlement of the megalopal stage on suitable substratum followed by metamorphosis into the first juvenile stage. Reducing the time to metamorphosis may result in higher recruitment and survival. Previous work has shown that metamorphosis of the Asian shore crab is accelerated by cues from three different sources: (a) water-soluble exudate produced by conspecific adult crabs; (b) biofilm covering rocks in natural habitat for this species; and (c) abiotic rock from natural habitat. The objective of the present investigation was to characterize the metamorphic cue associated with biofilm from rocky intertidal habitat and to compare the three metamorphic cues (exudate from conspecific adults, biofilm from rocky intertidal, and texture of substratum) that have been identified for H. sanguineus. Results of our study show that megalopae of the Asian shore crab respond strongly to biofilm associated with rocky intertidal habitat that has developed for at least 8 days. We also found that megalopae respond to textured rock surfaces from natural habitat, even when those surfaces had been rendered abiotic. The cue remains active after the biofilm has been exposed to − 20 ºC for 12 h, but is de-activated by a few minutes exposure to 100 °C. Moreover, the biofilm cue appears to work in synergy with cues from other sources, but requires actual contact with the biofilm. Our findings show that addition of biofilm to an abiotic textured rock surface significantly decreases mean time to metamorphosis, and simultaneous exposure of megalopae to biofilm-covered rock and to exudate from adult H. sanguineus decreases mean time to metamorphosis even further. The response of this species to multiple cues—and particularly to biofilm in the absence of adult conspecifics—provides a clear advantage in the colonization of virgin habitat and helps explain the very rapid spread of this invasive species along the majority of the east coast of the United States in only two decades.     

Characterization of the conspecific metamorphic cue for Hemigrapsus sanguineus (De Haan)

The Asian shore crab, Hemigrapsus sanguineus, is one of the most abundant invasive crabs along the east coast of the United States. Larval stages are generally planktonic, but the megalopa stage settles to the substratum near the time of metamorphosis. Reducing the time to metamorphosis may result in higher recruitment and survival. Previous work has shown that a water-soluble cue produced by adult H. sanguineus can induce metamorphosis of conspecific megalopae. Here we report the results of experiments in which megalopae were exposed to cues produced by different life stages of H. sanguineus. We also provide data from experiments that investigated the temporal stability, detection threshold, and chemical classification of the cue. Our results indicate that an active cue is produced by juveniles as well as adults. The cue is proteinaceous and begins to degrade within 2 days of production. The threshold for detection of the cue by megalopae lies between 0.1 and 0.01 µg of protein per ml.     

Trophic interactions between two introduced suspension-feeders, Crepidula fornicata and Crassostrea gigas, are influenced by seasonal effects and qualitative selection capacity

The effects of season and qualitative selection capacity on trophic relationships between two sympatric invasive suspension-feeders, Crepidula fornicata and Crassostrea gigas, were investigated in Bourgneuf Bay (France) from January 2003 to June 2004. Carbon and nitrogen stable isotopic deviations, δ¹³C and δ¹⁵N, of common Atlantic slippersnails and Pacific oysters were analysed relative to isotopic composition and availability of end-members.Slippersnail deviations were less variable over the sampling period compared with those of oysters. Significant differences between δ¹³C and δ¹⁵N of C. fornicata and C. gigas were found from winter to early summer, and linked to major isotopic changes in oysters. We identified three distinct seasonal periods: January to March when oysters were ¹⁵N-enriched compared to slippersnails and to themselves at other times of the year, April to June-July when oysters showed a ¹⁵N-depletion and a more marked ¹³C-depletion compared to slippersnails and to themselves at other times of the year, and July-August to December when both species presented similar carbon and nitrogen deviations. Species-specific differences in qualitative selection capability may explain these seasonal differences in isotopic deviations. Whereas the isotopic composition of the indiscriminate suspension-feeding slippersnails reflects the composition of the seston throughout the year, the oyster is capable of qualitative selection. The oyster isotopic compositions are consistent with a facultative activation of selection mechanisms under conditions of qualitative and quantitative food limitation, notably the preferential ingestion and assimilation of the dominant organic source in the suspended pool.We conclude that C. fornicata and C. gigas are trophic competitors only in winter and spring at this site, where detrital end-members are major POM components. These results underscore (1) the importance of long-term (annual) studies in the evaluation of potential trophic competition, and (2) the necessity to include the qualitative selection capacities of suspension-feeders in future interpretations of trophic relationships in marine coastal ecosystems.     

Differences between larval and pupal hemocytes of the tobacco hornworm, Manduca sexta, determined by monoclonal antibodies and density centrifugation

Insect hemocytes play a major role in developmental processes where they disassociate and rebuild metamorphosing tissues while undergoing physiological changes themselves. We identified hemocyte changes from the last larval to the beginning of the pupal stage of the tobacco hornworm, Manduca sexta. Larval and pupal hemocytes behaved differently in a 40% Percoll density gradient. Larval granular cells were found in almost all density layers, pupal granular cells were abundant in high density layers; larval plasmatocytes occurred in dense layers, pupal plasmatocytes became enriched in less dense layers of the gradient. Using a panel of monoclonal antibodies generated against purified hemocytes, several different antibody binding patterns were identified. Quantitative differences in staining intensities were observed more often than qualitative changes, e.g. a loss or a gain of staining. Both phenomena were related to both plasmatocytes and granular cells. The distribution of the corresponding antigens in tissues was tested on cross sections of larvae and pupae as well as in Western blot analyses using organ homogenates. Several antibodies were specific for hemocytes only, among which two antibodies bound to molecules of the hematopoietic organ. Other antibodies had an additional reactivity to other tissues, mainly to the basal lamina.     

Gene expression analysis of monospecies Salmonella typhimurium biofilms using differential fluorescence induction

Bacterial biofilm formation is an important cause of environmental persistence of food-borne pathogens, such as Salmonella Typhimurium. As the ensemble of bacterial cells within a biofilm represents different physiological states, even for monospecies biofilms, gene expression patterns in these multicellular assemblages show a high degree of heterogeneity. This heterogeneity might mask differential gene expression that occurs only in subpopulations of the entire biofilm population when using methods that average expression output. In an attempt to address this problem and to refine expression analysis in biofilm studies, we used the Differential Fluorescence Induction (DFI) technique to gain more insight in S. Typhimurium biofilm gene expression. Using this single cell approach, we were able to identify 26 genetic loci showing biofilm specific increased expression. For a selected number of identified genes, we confirmed the DFI results by the construction of defined promoter fusions, measurement of relative gene expression levels and construction of mutants. Overall, we have shown for the first time that the DFI technique can be used in biofilm research. The fact that this analysis revealed genes that have not been linked with Salmonella biofilm formation in previous studies using different approaches illustrates that no single technique, in casu biofilm formation, is able to identify all genes related to a given phenotype.     

Regulatory roles of nitric oxide during larval development and metamorphosis in Ciona intestinalis

Metamorphosis in the ascidian Ciona intestinalis is a very complex process which converts a swimming tadpole to an adult. The process involves reorganisation of the body plan and a remarkable regression of the tail, which is controlled by caspase-dependent apoptosis. However, the endogenous signals triggering apoptosis and metamorphosis are little explored. Herein, we report evidence that nitric oxide (NO) regulates tail regression in a dose-dependent manner, acting on caspase-dependent apoptosis. An increase or decrease of NO levels resulted in a delay or acceleration of tail resorption, without affecting subsequent juvenile development. A similar hastening effect was induced by suppression of cGMP-dependent NO signalling. Inhibition of NO production resulted in an increase in caspase-3-like activity with respect to untreated larvae. Detection of endogenously activated caspase-3 and NO revealed the existence of a spatial correlation between the diminution of the NO signal and caspase-3 activation during the last phases of tail regression. Real-time PCR during development, from early larva to early juveniles, showed that during all stages examined, NO synthase (NOS) is always more expressed than arginase and it reaches the maximum value at late larva, the stage immediately preceding tail resorption. The spatial expression pattern of NOS is very dynamic, moving rapidly along the body in very few hours, from the anterior part of the trunk to central nervous system (CNS), tail and new forming juvenile digestive organs. NO detection revealed free diffusion from the production sites to other cellular districts. Overall, the results of this study provide a new important link between NO signalling and apoptosis during metamorphosis in C. intestinalis and hint at novel roles for the NO signalling system in other developmental and metamorphosis-related events preceding and following tail resorption.     

Effects of salinity on larval stages of the rhizocephalan barnacle Loxothylacus texanus: survival and metamorphosis in response to the host, Callinectes sapidus

It is known that the rhizocephalan barnacle Loxothylacus texanus infects the greater blue crab, Callinectes sapidus, in the Gulf of Mexico and adjacent waters, however, factors that affect the prevalence and distribution of this parasite, particularly the dispersive larval stages of this organism, are not well understood. In the current study, the effects of salinity on larval survival and the metamorphosis of L. texanus in response to postmolt host exoskeleton were examined. Acute and acclimated responses were similar. Larval survival was highest in the 20-35‰ range, with 100% mortality of nauplii at all salinities <20‰ and >50‰. L. texanus cyprids were able to metamorphose over a broad range of salinities (15-60‰). In several cases, metamorphosis was actually greatest at high salinities (40-50‰). These data predict that L. texanus larvae would be concentrated in portions of Gulf of Mexico waters with salinities >20‰ such as the mouths of estuaries and bays. Conversely, upper regions of estuaries may be inhospitable to the dispersive (naupliar) stage of the parasite and may serve as a refuge from infection for host crabs.     

Ecdysteroids and juvenile hormones of whiteflies, important insect vectors for plant viruses

Ecdysteroids and juvenile hormones (JHs) regulate many physiological events throughout the insect life cycle, including molting, metamorphosis, ecdysis, diapause, reproduction, and behavior. Fluctuation of whitefly ecdysteroid levels and the identity of the whitefly molting hormone (20-hydroxyecdysone) have only been reported within the last few years. An ecdysteroid commitment peak that is associated with the reprogramming of tissues for a metamorphic molt in many holometabolous and some hemimetabolous insect species was not observed in last nymphal instars of either the sweet potato whitefly, Bemisia tabaci (Biotype B), or the greenhouse whitefly, Trialeurodes vaporariorum. Ecdysteroids reach peak levels 1-2 days prior to the initiation of the nymphal-adult metamorphic molt. Adult eye and wing differentiation which signal the onset of this molt begin earlier in 4th instar T. vaporariorum (Stages 4 and 5, respectively) than in B. tabaci (Stage 6), and the premolt peak is 3-4 times greater in B. tabaci ( approximately 400 fg/microg protein) than in T. vaporariorum ( approximately 120 fg/microg protein). The JH of B. tabaci nymphs and eggs was found to be JH III, supporting the view that JHs I and II are, with rare exception, only present in lepidopteran insects. In B. tabaci eggs, JH levels were approximately 10 times greater on day 2/3 (0.44 fg/egg or 0.54 ng/g) than on day 5 (0.04 fg/egg or 0.054 ng/g) post-oviposition. Approximately, 1.4 fg/2nd-3rd instar nymph (0.36 ng/g) was detected. It is probable that the relatively high level of JH in day 2/3 eggs is associated with the differentiation of various whitefly tissues during embryonic development.     

Immunocompetence of Galleria mellonella: sex- and stage-specific differences and the physiological cost of mounting an immune response during metamorphosis

The antibacterial immune response of the wax moth, Galleria mellonella, was analysed by use of an inhibition zone plate assay. We demonstrated significant stage-specific differences as the immune response was most effective in the pupal, next the larval and then the adult stage. In addition, we demonstrated that an immune challenge at the onset of, or during metamorphosis does not increase nor decrease the strength of the antibacterial immune response in the subsequent developmental stage(s). These findings illustrate that induced immunity is not preserved during metamorphosis but also deny any cost to the immune system itself. However, an immune challenge does induce a significant shortening of the direct development time and affects the mass loss during metamorphosis in a sex-dependent manner: males emerged smaller whereas the mass of females was not significantly affected. These observations indicate that there are sex-specific costs to mounting an immune response during metamorphosis which affect physiological traits, implicating a trade-off between immunity and development.     

Field reproductive dynamics of the invasive slipper limpet, Crepidula fornicata

At least part of the invasive success of the slipper limpet, Crepidula fornicata, in European waters must be due to reproductive characteristics, yet the events underlying the easily-observed brooding and non-brooding periods have not yet been studied in this species. The reproductive system dynamics were therefore investigated using topological histology and quantitative histological techniques. Specimens were sampled twice monthly for 18 months from Bourgneuf Bay, France, a mid-latitudinal point in the European distribution of C. fornicata. Both the testicles and ovaries showed active and resting phases, corresponding to the brooding and non-brooding periods, respectively. Maximum spermatozoan production corresponds to the female brooding period (female incubation of oviposited eggs, mid-March to late August), and allows males to possess full spermatozoan stocks at the height of fresh mature oocyte availability. The year-round presence of mature oocytes in the female gonad is misleading, since the histological aspect reveals that they are vestigial oocytes which slowly degenerate during the brooding period, possibly providing metabolites for the developing oocytes that become increasingly abundant during this period. A complete scheme of the C. fornicata reproductive cycle is presented, showing the events in the major reproductive organs.The seminal vesicle shows high inter-month variability in sperm presence, suggesting year-round copulation and sperm storage in the seminal receptacle. The seminal receptacle shows a uniform covering of spermatozoa throughout the year, suggesting rapid renewal after fertilization, again in line with multiple copulation throughout the year. Given the limited available space on the seminal epithelium, against which all spermatozoa abut, as well as polyandrous copulation, it is postulated that sperm competition may take place.     

Effects of quorum sensing on cell viability in Streptococcus mutans biofilm formation

Streptococcus mutans (S. mutans) uses a quorum sensing (QS) signaling system, which is dependent on competence stimulating peptide (CSP), to regulate diverse physiological activities including bacteriocin production, genetic transformation, and biofilm formation. However, the mechanism of the QS system-induced biofilm formation remains unclear. Here, we demonstrated that the late-stage biofilm formation was increased by the addition of exogenous CSP in S. mutans. The numbers of dead cells in biofilms formed in presence of CSP was 64.5% higher than that without CSP after 12 h (p < 0.05) and 76.3% higher after 24 h (p < 0.05), the numbers of live cells in biofilms formed in presence of CSP were 89.3% higher than that without CSP after 24 h (p < 0.01). The expression of QS-associated genes was increased 3.4-5.3-fold by CSP in biofilms. Our results revealed that cell viability of S. mutans grown in biofilms is affected by the CSP-dependent QS system.     

Biofilm formation by five species of Candida on three clinical materials

Most recalcitrant infections are associated with colonization and microbial biofilm development. These biofilms are difficult to eliminate by the immune response mechanisms and the current antimicrobial. Fungi can form biofilms on biomaterials commonly used in clinical practice (intravascular catheters, dentures, heart valves, implanted devices, contact lenses and other devices) and are associated with infections.A variety of in vitro models using different substrates/devices have been described. These models have been used to investigate the effect of different variables, including flow, growth time, nutrients and physiological conditions on fungal biofilm formation, morphology and architecture.The purpose of our study is to analyze biofilm formation capacity by 84 strains of Candida spp. (23 C. albicans, 23 C. parapsilosis, 16 C. tropicalis, 17 C. glabrata and 5 C. krusei) on three materials used in medical devices and its quantification using a method based on viable cell count.Under the conditions of our study, all assayed Candida strains have been able to form biofilms. All species showed greater biofilm formation capacity on Teflon™, with the exception of C. glabrata which displayed higher biofilm formation capacity on PVC. Biofilm formation by Candida spp. varies depending on the type of material on which it grows and on the species and strain of Candida.The method we propose could be of great use to deepen scientific knowledge on this subject of remarkable clinical significance, considering the absence of standard biofilm formation and quantification techniques on the catheters and the level of difficulty associated to those available.     

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.     

Metamorphosis in a semiterrestrial crab, Sesarma curacaoense: intra- and interspecific settlement cues from adult odors

The larvae of many marine invertebrate species are able to delay their settlement and metamorphosis in the absence of characteristic cues from the adult habitat. This phenomenon was experimentally studied in the megalopa stage of Sesarma curacaoense de Man, 1892, a semiterrestrial grapsid crab that lives in the shallow coastal mangrove habitats in the Caribbean region. Duration of the development and survival to metamorphosis to the first juvenile crab stage were compared between experimental treatments, where the water was conditioned with adult crabs (“adult-conditioned water,” ACW) and control groups reared in filtered seawater. In the experiments with larvae from two different females, development duration was significantly shorter and mortality lower in water conditioned with conspecific adults. In the two control groups, the effects of supply with an artificial substrate (nylon gauze) were tested. This comparison showed that the presence of substrate did not significantly influence the time to metamorphosis, but did reduce the mortality rate. In all later experiments, the megalopae were thus routinely provided with nylon gauze as a substrate. In each of the three subsequent replicate experiments conducted with larvae from different females, survival rate and development time to metamorphosis were compared between one control group and four treatments with ACW. The effectiveness of conspecific (S. curacaoense) adult odors as metamorphosis-stimulating cue was, in these experiments, compared with that of ACW from one congener (S. rectum) and two species belonging to different genera within the Grapsidae (Armases miersii, Chasmagnathus granulata). While the rate of survival showed inconsistent patterns among repeated experiments, the development was consistently fastest with conspecific ACW, followed by ACW from S. rectum, A. miersii and C. granulata. Only the conspecific and congeneric cues had statistically significant effects (i.e. shorter development than in the controls). These response patterns suggest that chemically similar factors (presumably pheromones) are produced by closely related species and, thus, their chemical structure may reflect phylogenetical relationships within a clade.     

Trypanosoma cruzi: ultrastructural studies of adhesion, lysis and biofilm formation by Serratia marcescens

A few days after blood meal the number of bacteria in the anterior midgut (stomach) of Rhodnius prolixus, a vector of Trypanosoma cruzi, the causative agent of Chagas' disease, increases dramatically. Many of the bloodstream trypomastigotes of the pathogenic protozoan as well as ingested erythrocytes are lysed in the stomach. Incubation of T. cruzi with Serratia marcescens variant SM365, lead to parasite lysis. In the present study, this bacterium rapidly adhered to the protozoan surface through d-mannose recognizing fimbriae and rapidly induced its complete lysis. In contrast, the DB11 variant of the same bacterial species did not adhere and did not induce protozoan lysis. Scanning and transmission electron microscopy revealed that following bacteria-protozoan attachment there is an assembly of long filamentous structures, identified as a biofilm, which connect the protozoan to the bacteria forming bacterial clusters. We conclude that parasite lysis and biofilm formation mechanisms are important for understanding parasite-microbiota interactions in the gut of insect vectors of trypanosomatids.     

Barnacle larval destination: piloting possibilities by bacteria and lectin interaction

Modulation of metamorphosis in barnacles in response to cues of biological origin is established. The bacteria associated with the barnacles also have a role in such modulations. We isolated the bacteria, Pseudomonas aeruginosa, Bacillus pumilus and Citrobacter freundii from the shell surface of Balanus amphitrite and assayed against its cypris larvae. The former species was promotory while the latter two inhibited cyprid metamorphosis. P. aeruginosa however, when tagged with lectins specific to glucose and its derivatives, mannose and fructofuranose negated the promotory effect. Whereas, tagging of galactose derivatives translated the inhibitory effect of B. pumilus and C. freundii into a promotory one showing that lectins can alter the signals in either direction. Galactose-binding lectins have been identified in the haemolymph of barnacles, which could find their way through the excretory system to the surface. The presence of such lectins could probably provide this organism with an ability to alter the signals or cues. Microscale patchiness of bacteria is also evident on surfaces in the sea. The availability of conflicting cues in patches may help pilot the larvae to their settlement destination. Understanding these controlling mechanisms and interfering with the pathways that are involved in lectin synthesis would be a step forward in antifouling technology.     

The Him gene inhibits the development of Drosophila flight muscles during metamorphosis

During Drosophila metamorphosis some larval tissues escape the general histolysis and are remodelled to form adult tissues. One example is the dorso-longitudinal muscles (DLMs) of the indirect flight musculature. They are formed by an intriguing process in which residual larval oblique muscles (LOMs) split and fuse with imaginal myoblasts associated with the wing disc. These myoblasts arise in the embryo, but remain undifferentiated throughout embryogenesis and larval life, and thus share characteristics with mammalian satellite cells. However, the mechanisms that maintain the Drosophila myoblasts in an undifferentiated state until needed for LOM remodelling are not understood. Here we show that the Him gene is expressed in these myoblasts, but is undetectable in developing DLM fibres. Consistent with this, we found that Him could inhibit DLM development: it inhibited LOM splitting and resulted in fibre degeneration. We then uncovered a balance between mef2, a positive factor required for proper DLM development, and the inhibitory action of Him. Mef2 suppressed the inhibitory effect of Him on DLM development, while Him could suppress the premature myosin expression induced by mef2 in myoblasts. Furthermore, either decreased Him function or increased mef2 function disrupted DLM development. These findings, together with the co-expression of Him and Mef2 in myoblasts, indicate that Him may antagonise mef2 function during normal DLM development and that Him participates in a balance of signals that controls adult myoblast differentiation and remodelling of these muscle fibres. Lastly, we provide evidence for a link between Notch function and Him and mef2 in this balance.     

Delineating metamorphic pathways in the ascidian Ciona intestinalis

In most ascidians, metamorphosis of tadpole-like swimming larvae is accompanied by dynamic changes in their shape to form sessile adults. The mechanisms underlying ascidian metamorphosis have been debated for a long time. Although recent molecular studies have revealed the presence of various molecules involving in this process, the basic mechanism of the metamorphic events is still unclear. For example, it has not been solved whether all metamorphic events are organized by the same single pathway or by multiple, independent pathways. In the present study, we approached this question using the ascidian Ciona intestinalis. When the papillae and preoral lobes of the larvae were cut off, the papillae-cut larvae initiated certain trunk metamorphic events such as the formation of an ampulla, body axis rotation and adult organ growth without other metamorphic events. This observation indicates that metamorphic events can be divided into at least two groups, events initiated in the papillae-cut larva and events not initiated in this larva. In addition to this observation, we have isolated a novel mutant, tail regression failed (trf), which shows similar phenotypes to those of papillae-cut larvae. The phenotypes of trf mutants are basically different from those of swimming juvenile mutants (Sasakura, Y., Nakashima, K., Awazu, S., Matsuoka, T., Nakayama, A., Azuma, J., Satoh, N., 2005. Transposon-mediated insertional mutagenesis revealed the functions of animal cellulose synthase in the ascidian Ciona intestinalis. Proc. Natl. Acad. Sci. U. S. A. 102, 15134-15139.), which also show abnormal metamorphosis. These findings suggest a model by which ascidian metamorphic events can be classified into four groups initiated by different pathways.