Larval settlement and metamorphosis in the slipper limpet Crepidula onyx (Sowerby) in response to conspecific cues and the cues from biofilm

In the marine environment, aggregated distribution in the genus Crepidula is a very common phenomenon. Works from Pechenik's group suggested that this is the result of gregarious settlement of larvae in response to cues associated with conspecific adults. In this study, we investigated the existence of larval metamorphic cues associated with adults of C. onyx, a slipper limpet introduced to Hong Kong from the U.S. in the 1970s, through a series of laboratory bioassays. The results showed that derived cues in adult C. onyx were waterborne and the waterborne cues were not derived from bacteria associated with the shell and soft body of the adult Crepidula. The natural biofilm also induced the larval metamorphosis of C. onyx. The cues from the biofilm were associated with the surface of the biofilm and were not waterborne. The aggregated distribution in nature of adult C. onyx may result from a selective larval settlement process. On a small scale in the water column near the conspecific adults, larvae of C. onyx initially detect the waterborne conspecific cues, which then lead to positive downward swimming or passive sinking. This activity increases the chances for larvae to make contact with the biofilm and to be exposed into the higher concentration of waterborne conspecific cues. This may eventually lead to the enhanced larval settlement pattern on or near the conspecific adults.     

Multiple cues from multiple habitats: Effect on metamorphosis of the Florida stone crab, Menippe mercenaria

The Florida stone crab, Menippe mercenaria, is an economically and ecologically important species that ranges from North Carolina throughout the Caribbean and the southeastern Gulf of Mexico. However, there is little known about its early life history stages as compared to other commercially important species in the region. The goal of this research was to examine effects of putative cues on metamorphosis from the megalopa stage to the first juvenile stage. Our study investigated the effect of water-soluble exudates from four substrata, as well as natural biofilms, and exudates from adult stone crabs. In addition, the influence of natural substrata was compared to that of artificial substrata. Adult exudate had no significant effect on metamorphosis, despite a wide range of tested concentrations. In contrast, there was a significant effect on mean time to metamorphosis in experimental groups exposed to multiple cues associated with the brown alga Sargassum fluitans, rubble from stone crab habitat, the eastern oyster Crassostrea virginica, and biofilms associated with the oyster. Furthermore, we provide evidence for metamorphic responses to water-soluble chemical cues, as well as biochemical and physical cues associated with different substrata. Overall results were coherent with the relevant body of previous work on metamorphosis of brachyuran crab larvae and indicate that both physical and chemical cues are important factors in facilitating the settlement and metamorphosis of M. mercenaria larvae in juvenile nursery habitat.     

Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella

Infection of Galleria mellonella by feeding a mixture of Bacillus thuringiensis spores or vegetative bacteria in association with the toxin Cry1C results in high levels of larval mortality. Under these conditions the toxin or bacteria have minimal effects on the larva when inoculated separately. In order to evaluate whether G. mellonella can function as an oral infection model for human and entomo-bacterial pathogens, we tested strains of Bacillus cereus, Bacillus anthracis, Enterococcus faecalis, Listeria monocytogenes, Pseudomonas aeruginosa and a Drosophila targeting Pseudomonas entomophila strain. Six B. cereus strains (5 diarrheal, 1 environmental isolate) were first screened in 2nd instar G. mellonella larvae by free ingestion and four of them were analyzed by force-feeding 5th instar larvae. The virulence of these B. cereus strains did not differ from the B. thuringiensis virulent reference strain 407Cry⁻ with the exception of strain D19 (NVH391/98) that showed a lower virulence. Following force-feeding, 5th instar G. mellonella larvae survived infection with B. anthracis, L. monocytogenes, E. faecalis and P. aeruginosa strains in contrast to the P. entomophila strain which led to high mortality even without Cry1C toxin co-ingestion. Thus, specific virulence factors adapted to the insect intestine might exist in B. thuringiensis/B. cereus and P. entomophila. This suggests a co-evolution between host and pathogens and supports the close links between B. thuringiensis and B. cereus and more distant links to their relative B. anthracis.     

Relevance of biofilm bacteria in modulating the larval metamorphosis of Balanus amphitrite

Natural microbial communities found on different substrata exposed to the marine environment, including barnacle shell surfaces, are reported to have varying influences on the settlement and metamorphosis of competent cypris larvae. Experiments were carried out to compare the influence of settlement-inducing compounds from the bacteria isolated from the shell surface of Balanus amphitrite on its larval metamorphosis. The effect of multispecies bacterial film was also assessed. The production of different molecules by the bacteria was influenced by the nutrient media under which they were grown. It was observed that the promotory multispecies bacterial film turned to inhibition mode in the presence of the adult extract of the barnacle, indicating that bacteria-adult extract interactions alter the synthesis of different compounds produced by bacteria. The studies also show that the waterborne and the surface-associated cues from the bacteria function differentially in mediating larval metamorphosis. Understanding the complexities involved in such interactions and identification of the factors governing them would be a step forward.     

Low salinity stress experienced by larvae does not affect post-metamorphic growth or survival in three calyptraeid gastropods

Marine larvae that experience some sub-lethal stresses can show effects from those stresses after metamorphosis, even when they seem to recover from those stresses before metamorphosis. In this study we investigated the short and long-term effects of exposing the larvae of three calyptraeid gastropods (Crepidula fornicata, Crepidula onyx, and Crepipatella fecunda) to temporary reductions in salinity. Larvae of all three species showed slower larval growth rates, longer time to metamorphic competence, and substantial mortality after being stressed in seawater at salinities of 10, 15, and 20 for less than 48 h. Larval tolerance to low salinities varied widely within and among species, but longer stresses at lower salinities were generally more harmful to larvae. However, larvae in nearly all experiments that were able to metamorphose survived and grew normally as juveniles; there were no documented “latent effects.” For all three species, starving larvae in full-strength seawater was not as harmful as exposing larvae to low salinity stress, indicating that detrimental effects on larvae were caused by the salinity stress per se, rather than by an indirect effect of salinity stress on feeding. C. fornicata that were stressed with low salinity as juveniles were more tolerant of the stress than larvae: all stressed juveniles lived and showed reduced growth rates for no more than 3 days. Our data suggest that even though reduced salinity is clearly stressful to the larvae of these 3 gastropod species, metamorphosis seems to generally provide individuals with a fresh start.     

The effect of bacterial and diatom biofilms on the settlement of the bryozoan Bugula neritina

Biofilms of marine bacteria and diatoms and their combinations were examined in laboratory choice assays to determine their effects on the attachment and successful metamorphosis of the larvae of the bryozoan Bugula neritina (Linnéus). The larval settlement in response to unfilmed surfaces, a natural biofilm (NBF) and adsorbed cells of three strains of bacteria, five strains of pennate diatoms and combinations of the two at different densities. Bacterial and diatom strains showed different effects on the larval settlement of B. neritina. Bacterial monospecific strains of an unidentified α-Proteobacterium and Vibrio sp. mediated the same percentage of settlement as a filtered seawater control. Biofilms of Pseudoalteromonas sp. caused significantly lower larval settlement. Larval settlement of B. neritina was negatively correlated with increasing densities of Pseudoalteromonas sp. The highest percentages of settlement were mediated by the biofilms of the diatom species Achnanthes sp., Amphora cofeaeformis, Amphora tenerrima, Nitzschia constricta and a 5-day-old natural biofilm, while the lowest settlement was found on a N. frustulum film. A three-way analysis of variance demonstrated that the density of bacteria and the presence of particular species of diatoms and bacteria in combined biofilms, significantly affected the settlement of B. neritina larvae. High settlement of larvae (50-90%) at all treatments indicated that B. neritina larvae are much more indiscriminate settlers than previously expected. Hence, using this species as a monitoring organism to trace ecologically relevant subtle changes of settlement cues in the natural environment should be carefully re-examined.     

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.     

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.     

Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm

We examined interaction between Bacillus thuringiensis subsp. kurstaki HD-1 (Foray 48B) and larval midgut bacteria in two lepidopteran hosts, Lymantria dispar and Choristoneura fumiferana. The pathogen multiplied in either moribund (C. fumiferana) or dead (L. dispar) larvae, regardless of the presence of midgut bacteria. Inoculation of L. dispar resulted in a pronounced proliferation of enteric bacteria, which did not contribute to larval death because B. thuringiensis was able to kill larvae in absence of midgut bacteria. Sterile, aureomycin- or ampicillin-treated larvae were killed in a dose-dependent manner but there was no mortality among larvae treated with the antibiotic cocktail used by [Broderick et al., 2006] and [Broderick et al., 2009]. These results do not support an obligate role of midgut bacteria in insecticidal activity of HD-1. The outcome of experiments on the role of midgut bacteria may be more dependent on which bacterial species are dominant at the time of experimentation than on host species per se. The L. dispar cohorts used in our study had a microflora, that was dominated by Enterococcus and Staphylococcus and lacked Enterobacter. Another factor that can confound experimental results is the disk-feeding method for inoculation, which biases mortality estimates towards the least susceptible portion of the test population.     

Optimal foraging versus shared doom effects: interactive influence of mussel size and epibiosis on predator preference

In the western Baltic Sea, the highly competitive blue mussel Mytilus edulis tends to monopolize shallow water hard substrata. In many habitats, mussel dominance is mainly controlled by the generalist predator Carcinus maenas. These predator-prey interactions seem to be affected by mussel size (relative to crab size) and mussel epibionts.There is a clear relationship between prey size and predator size as suggested by the optimal foraging theory: Each crab size class preferentially preys on a certain mussel size class. Preferred prey size increases with crab size.Epibionts on Mytilus, however, influence this simple pattern of feeding preferences by crabs. When offered similarly sized mussels, crabs prefer Balanus-fouled mussels over clean mussels. There is, however, a hierarchy of factors: the influence of attractive epibiotic barnacles is weaker than the factor ‘mussel size’. Testing small mussels against large mussels, presence or absence of epibiotic barnacles does not significantly alter preferences caused by mussel size. Balanus enhanced crab predation on mussels in two ways: Additional food gain and, probably more important, improvement in handling of the prey. The latter effect is illustrated by the fact that artificial barnacle mimics increased crab predation on mussels to the same extent as do live barnacles.We conclude that crab predation preferences follows the optimal foraging model when prey belong to different size classes, whereas within size classes crab preferences is controlled by epibionts.     

Polystoma gallieni: Experimental evidence for chemical cues for developmental plasticity

Among monogeneans that display direct life cycles, plastic developmental strategies may have been selected to counter the lack of transmission opportunities. Within amphibian polystomatids, some species of the genus Polystoma develop into two different phenotypes depending on the host physiological stage to which free swimming larvae attach. When oncomiracidia infest old tadpoles, they develop slowly and migrate during host metamorphosis towards the bladder where they reach maturity. On the other hand when larvae infest young tadpoles, they develop rapidly into neotenic phenotypes that reproduce in the branchial chamber. These alternative developments are explored through experimental infestations with Polystoma gallieni, the specific polystome of the stripeless tree frog Hyla meridionalis. When oncomiracidia were left for 6 h in water in which young tadpoles had been previously maintained for one day, they mainly developed into the neotenic phenotype regardless of the tadpole stage they encountered later. This indicates that P. gallieni could collect released host-derived signals before physical contact with its host.     

The chemical cue tetrabromopyrrole from a biofilm bacterium induces settlement of multiple Caribbean corals

Microbial biofilms induce larval settlement for some invertebrates, including corals; however, the chemical cues involved have rarely been identified. Here, we demonstrate the role of microbial biofilms in inducing larval settlement with the Caribbean coral Porites astreoides and report the first instance of a chemical cue isolated from a marine biofilm bacterium that induces complete settlement (attachment and metamorphosis) of Caribbean coral larvae. Larvae settled in response to natural biofilms, and the response was eliminated when biofilms were treated with antibiotics. A similar settlement response was elicited by monospecific biofilms of a single bacterial strain, Pseudoalteromonas sp. PS5, isolated from the surface biofilm of a crustose coralline alga. The activity of Pseudoalteromonas sp. PS5 was attributed to the production of a single compound, tetrabromopyrrole (TBP), which has been shown previously to induce metamorphosis without attachment in Pacific acroporid corals. In addition to inducing settlement of brooded larvae (P. astreoides), TBP also induced larval settlement for two broadcast-spawning species, Orbicella (formerly Montastraea) franksi and Acropora palmata, indicating that this compound may have widespread importance among Caribbean coral species.     

Investigation of biota associated and natural infection by sporulated bacteria in Culicidae larvae from São Paulo state, Brazil

The aim of this study was to investigate the naturally occurring bacteria from Culicidae larvae found in São Paulo state between 2006 and 2008 from the Tiete Ecological Park in the municipality of Caraguatatuba. Bacterial strains were obtained after surface sterilization of larvae followed by thermal treatment and incubation in bacteriological media under laboratory conditions. Identification was determined using cytomorphology, biochemical and physiological tests. Strains were characterized by qualitative evaluation of biological activity against Culicidae larvae, comparing protein profiles obtained by electrophoresis of crystal protoxins, electrophoresis of Lysinibacillus sphaericus isoenzymes, detection of the L₂ fraction of Hemolytic BL enterotoxin and amplification of DNA using Multiplex-PCR to detect HBL, NHE, CytK, BceT and EntFM enterotoxins. The results obtained in this study demonstrated that the endosymbiotic bacterial diversity belonging to Bacillus and related genera associated with Culicidae larvae is limited to a number of species that does not vary throughout the seasons.     

The role of epibotic bacteria from the surface of the soft coral Dendronephthya sp. in the inhibition of larval settlement

It has been suggested that bacteria associated with soft-bodied organisms are suggested to produce bioactive compounds against the attachment of invertebrate larvae and bacteria onto the surface of these organisms. Our recent study has demonstrated that epibiotic bacteria from the surface of the soft coral Dendronephthya sp. (Coelenterata: Octocoralia, Alcyonacea) inhibit the growth of bacteria commonly found in marine natural biofilms. In the present study, the effect of 11 epibiotic bacteria isolated from the surface of Dendronephthya sp. on larval settlement of the tubeworms Hydroides elegans was examined using laboratory bioassay. Among 11 bacterial isolates, 2 strains (18%) inhibited the larval settlement of H. elegans (Haswell), 4 strains (36%) were “inductive” to larvae and the remaining 5 strains (46%) were “non-inductive”. There was no correlation between the antifouling activities of bacterial isolates and their phylogenetic origin, i.e. closely related bacterial strains showed different effects on larval settlement of H. elegans. When all “inductive”, “non-inductive” and “inhibitive” bacterial isolates were mixed in a 1:1:1 ratio, the effect of the resultant multispecies film on larval settlement became “inhibitive”. Waterborne compounds of Vibrio sp. and an unidentified α-Proteobacterium, which suppressed the settlement of H. elegans and Bugula neritina (L.) larvae, were further investigated using size fractionation and bioassay-guided enzymatic analysis. It was found that antilarval settlement compounds from these bacteria were heat-stable polysaccharides with a molecular weight >100 kDa. The results indicate that the bacteria associated with the soft coral Dendronephthya sp. may contribute to the antifouling mechanisms of the soft-bodied organisms by producing compounds that are against bacterial growth and settlement of macrofoulers on the surface of their host.     

Structures of six cDNAs expressed specifically at cypris larvae of barnacles, Balanus amphitrite

We cloned six cDNAs by screening cDNA libraries of cypris larvae from barnacles, Balanus amphitrite, and studied their expression by Northern blot analysis. All of them are expressed in the cypris larvae at the settlement stage, but not in the earlier nauplii larvae nor in later adult barnacles. Therefore, we designated them as barnacle cypris larva-specific genes (bcs); bcs-1, bcs-2, bcs-3, bcs-4, bcs-5 and bcs-6. During the process of larval attachment and metamorphosis, the amounts of bcs-1 and bcs-2 mRNAs decreased, whereas the bcs-3, bcs-4, bcs-5 and bcs-6 mRNAs increased. A homology search showed that all cDNAs encode novel peptides containing characteristic amino acid sequences. This study strongly suggests that these bcs gene products are involved in the cypris larval attachment and metamorphosis of barnacles.     

Surface wettability as a determinant in the settlement of the barnacle Balanus Improvisus (DARWIN)

Several studies have shown that the initial surface wettability, is of importance in the settlement of macrofouling larvae such as barnacles, bryozoans and hydroids in the field as well as in laboratory assays. In this study we present results from laboratory assays using hydrophilic and hydrophobic polystyrene (PS) and cyprid larvae of Balanus improvisus (Darwin). The results obtained differ markedly from those reported for the barnacle Balanus amphitrite (Darwin), where a high surface wettability seemed to be preferred for settlement. Our results show that a surface with intermediary wettability (hydrophilic PS) reduced settlement by 38% as compared to surfaces of low wettability (hydrophobic PS) during an 8-day period. During the experiment, the wettability in the hydrophilic PS dishes was not significantly changed as measured by advancing contact angle with mQ water. Over an 8-day period wettability of the hydrophobic PS dishes approached that of the hydrophilic PS surfaces. We further conducted experiments with highly hydrophilic and highly hydrophobic methylsilane-treated glass surfaces with known chemistry. In this experiment, the settlement of cyprid larvae was completely inhibited by the high wettability surfaces. Contact angle measurements revealed that the wettability during the length of the experiment of the hydrophilic glass surfaces was not significantly altered. We conclude by these experiments that even an intermediate wettability can significantly affect the overall settlement success of the barnacle B. improvisus. The mechanism by which the settlement is impeded might be biologically mediated through the recognition by cyprid larvae of the molecular composition of the surface when the cyprid reverts to the settlement phase, i.e. when swimming behaviour is abandoned in favour of surface exploration, or it is mediated by physicochemical forces acting between the surface and the larval body or the larval antennules.     

Behavioral plasticity of the soft-shell clam, Mya arenaria (L.), in the presence of predators increases survival in the field

Soft-shell clams, Mya arenaria, are sessile, suspension-feeding bivalves that are preyed upon by the exotic green crab, Carcinus maenas. Clams evade crab consumers by burrowing deeper into the sediment after perceiving a threat from a nearby predator. The purpose of this study was to determine the types of signals that M. arenaria use to detect predators and the types of behaviors clams use to avoid being eaten. In a field study, clams increased their burial depth in the presence of green crab predators consuming conspecifics that were caged nearby, and also increased burial depth after artificial tactile stimulation in the laboratory assay. These results indicate that clams can use chemical and mechanical cues to detect potential predatory threats. We performed a field study to examine the difference in survivability of clams that had burrowed deeper into the sediment in response to predators vs. control clams that were burrowed less deeply. Significantly higher survival rates were observed in clams that had initially burrowed more deeply, suggesting that increasing burial depth is a valid predator avoidance strategy. Some bivalves also alter their pumping rates in the presence of predators, making them less apparent and providing more structural defense by covering soft tissue, and we measured pumping time of soft-shell clams in the presence and absence of predators, when burrowing was not an option for escape. Soft-shell clams did not alter their pumping time in the presence of green crab predators, possibly because they employ a burrowing method called “hydraulic” or “jet-propelled” burrowing, where it is necessary for the clam to pump in order to burrow. Chemical signals and tactile cues instigated behavioral changes in M. arenaria, and this change in behavior (increasing burial depth) increased clam survival in the field.     

Termiticidal activity of lectins from Myracrodruon urundeuva against Nasutitermes corniger and its mechanisms

The isolation of lectins from Myracrodruon urundeuva bark (MuBL) and heartwood (MuHL) as well as the termiticidal activity of MuHL against Nasutitermes corniger has already been described. This work reports on the purification of a leaf lectin (MuLL) and the characterization of MuBL, MuHL, and MuLL; also described are the resistance of hemagglutinating activity of the three lectins to trypsin activity from N. corniger gut and the termiticidal activity on N. corniger of MuBL (LC₅₀ of 0.974 mg ml⁻¹ on workers and 0.787 mg ml⁻¹ on soldiers) and MuLL (LC₅₀ of 0.374 mg ml⁻¹ on workers and 0.432 mg ml⁻¹ on soldiers). The antibacterial effect of MuBL, MuHL, and MuLL on bacteria from gut of N. corniger was also investigated and lectins showed similar bacteriostatic activity (MIC of 62.5 ??g ml⁻¹ for workers and 125 ??g ml⁻¹ for soldiers). MuBL and MuHL were more efficient bactericidal agents on bacteria in the workers’ gut (MBC of 125 ??g ml⁻¹) than MuLL (MBC of 250 ??g ml⁻¹) and similar bactericidal activity was detected on bacteria in the gut of soldiers (MBC of 250 ??g ml⁻¹). The termiticidal activity of M. urundeuva lectins can be explained by the chitin-binding property, resistance to termite digestive enzyme, and the antibacterial effect on symbiotic bacteria of N. corniger gut.     

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.     

Larvae of the ectoparasitic wasp,Eulophus pennicornis,release factors which adversely affect haemocytes of their host,Lacanobia oleracea

When larvae of the ectoparasitic wasp Eulophus pennicornis were incubated for 4 h on balls of cotton wool soaked in tissue culture medium (TC-100), they released a variety of factors. Subsequent incubation of these larval wasp secretions with monolayers of haemocytes from their host, Lacanobia oleracea, demonstrated that they adversely affect haemocyte morphology, behaviour and viability. For instance, when monolayers of haemocytes were incubated for 18 h in TC-100, approximately 73% of the cells present, attached firmly to and spread over the tissue culture surface by extending pseudopods. By contrast, when incubated in TC-100 containing larval wasp secretions, only about 27% of the haemocytes present remained attached to the tissue culture surface after washing. The majority of these had a rounded configuration and neither spread nor extended pseudopods. Furthermore, viability assays indicated that approximately 36% of the attached haemocytes were dead, as opposed to 11-12% in the controls. The E. pennicornis secretions also significantly reduced the ability of L. oleracea haemocytes to move across the surface of the slide and form clumps (p≤0.0005) and to phagocytose FITC-labelled Escherichia coli in vitro (p≤0.0005). These results indicate that secretions from E. pennicornis larvae contain an anti-haemocyte factor(s) that can kill and/or alter the behaviour of host haemocytes. As a result, the ability of the haemocytes to execute important immune responses is compromised. Preliminary data suggest that the active molecules are proteins, and that their mechanism of action may involve inhibition of polymerization and/or disorganization of the haemocyte cytoskeleton.