Bactericidal activity of hemolymph from the horseshoe crab, Limulus polyphemus

Bactericidal activity was found in Limulus serum, with great individual variation in titers toward different bacteria and also among individual horseshoe crabs toward the same bacterial species. These titers varied between monthly determinations of activity. There were crabs with zero activity toward each bacterial species tested. Although environmental factors are likely influences on the bactericidal activity of Limulus serum, the marked variability within similarly treated groups indicates large individual differences in the horseshoe crab population. The highest titers were recorded against those Gram-negative bacilli found normally in the environment. Lower titers were found against those species found normally in warmblooded animals and present in water as contaminants. The serum bactericidal factor is probably released from the circulating amoebocytes during clotting since there was no activity in the “plasma” portion of the blood. Exposure to heat (56°C, 30 min) destroyed the bactericidal activity.     

Lectins in the hemolymph of Japanese horseshoe crab, Tachypleus tridentatus

The hemolymph of the Japanese horsehoe crab, Tachypleus tridentatus contains lectins which agglutinate mammalian erythrocytes. Affinity chromatographic purification of the lectins using bovine submaxillary gland mucin-conjugated Sepharose resulted in the separation of the lectins into four fractions; one major and three minor lectins. Protein subunits revealed by polyacrylamide gel electrophoresis and the immunoprecipitin line of these lectins against antiserum to crude lectins were unique to each fraction. The activities of all the lectins were optimal at pH values between 6 and 8, and were destroyed by heating at 60°C. Calcium chloride augumented the activities of three lectins, but the major lectin was not influenced by the salt. Bovine erythrocytes were not agglutinated by any of the lectins and comparative agglutination titers for other erythrocytes from various sources were different among these lectins. The activities of all the lectins were inhibited by N-acetylamino sugars. They were more effectively inhibited by glycoproteins which contain sialic acid.     

Propagule predation in a Neotropical mangrove: the role of the Grapsid crab Goniopsis cruentata

In Neotropical mangroves the crabs Ucides cordatus and Goniopsis cruentata have been considered the most significant propagule consumers, but their relative importance has not been investigated. The aim of this study was to compare the magnitude of predation by these crabs on three mangrove species propagules: Avicennia schaueriana, Laguncularia racemosa and Rhizophora mangle. We found that G. cruentata is a more important predator than U. cordatus in both natural and restored areas. We also tested the hypothesis that Ucides and Goniopsis have antagonistic effects on propagules predation using a cage experiment where the presence/absence of these species was manipulated in a 2 × 2 factorial design. The effects of Goniopsis were stronger in the absence of Ucides due to negative interactions between these predator species. Moreover, we found that Goniopsis preference for A. schaueriana and L. racemosa can favor the dominance of R. mangle in Neotropical mangroves. This study suggests that propagule predation by Goniopsis should be controlled in mangrove restoration programs at abandoned shrimp farms and destroyed areas, if dominance by R. mangle is undesirable relative to mixed species communities.     

Mercury and osmoregulation in the euryhaline crab,Eriocheir sinensis

This review will focus on cases where it might be possible that the toxicity of the heavy metal mercury results from an interaction with osmoregulatory mechanisms. It is shown that mercury-induced impairment of osmoregulatory capability in the sense of severe modifications of the blood osmotic concentration is more pronounced in brachyuran decapod species adapted to dilute waters. The rationale for considering these effects is based on a comparison between mercury effects on three species of decapod crustaceans exhibiting various degrees of osmoregulation capability: the strong regulator crab Eriocheir sinensis, the weak regulator Carcinus maenas, both of them being euryhaline, and the stenohaline osmoconformer Cancer pagurus. It is established that a synergistic effect exists between salinity and HgCl₂ toxicity in euryhaline species which are hyperregulators in dilute media, that is, E. sinensis and C. maenas only. Depth study of E. sinensis as a model demonstrates that Na⁺ and Cl⁻ permeabilities of the gill epithelium is affected by mercury, as well as the Na⁺ and Cl⁻ active transport processes located at the same level. Evidences are brought showing that mercury drastically disturbs the Na⁺/K⁺ pump and the Cl⁻ channels located in the serosal baso-lateral membranes of the posterior gills.     

Interaction of chloroplasts with inhibitors: induction of chlorosis by diuron during prolonged illumination in vitro

A primary symptom of diuron (DCMU) phytotoxicity in plants is the destruction of chlorophyll. To study this process in vitro, chloroplasts from pea leaves (Pisum sativum L.) have been incubated in the light with DCMU for periods of up to 34 hours. The sequence of photodestruction of chlorophylls and carotenoids has been followed to try and establish the nature of the chloroplast protection mechanisms that are destroyed by DCMU. β-Carotene decays most rapidly, followed by chlorophyll a and xanthophylls which are destroyed in a constant ratio, followed finally by chlorophyll b. Bypassing the DCMU block in the electron transport system with an artificial electron donor provides complete protection against chlorophyll and carotenoid photodestruction. The same protection by this electron donor system is afforded to stroma-free lamellae from which soluble reductants have been removed so that NADPH formation, which has been proposed as an essential part of a protective xanthophyll cycle, is not possible. Both this and the simultaneous loss of chlorophyll a and xanthophylls tend to preclude the breakdown of a xanthophyll cycle from the possible protective mechanisms inhibited or destroyed by DCMU.     

The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense

Targett G. A. T. and Wilson V. C. L. C. 1973. The blood incubation infectivity test as a means of distinguishing between Trypanosoma brucei brucei and T. brucei rhodesiense. International Journal for Parasitology, 3: 5–11. A simple test for distinguishing between the morphologically identical subspecies Trypanosoma brucei rhodesiense, which is infective to man, and T. brucei brucei, which by definition is not, has been described. This test, the blood incubation infectivity test (BIIT), is based on absolute differences in the infectivity to rats of the subspecies after exposure to human blood, and was applied to strains which are preserved in the laboratory as stabilates. Five T. brucei brucei strains were BIIT negative since their infectivity was destroyed by incubation in normal human blood but only five of the nine T. brucei rhodesiense strains tested were consistently BIIT positive. The other four gave equivocal results, indicating that the resistance of T. brucei rhodesiense strains to the trypanocidal effect of human blood can change, probably as a result of maintenance in the laboratory.     

Tissue specificities of Thelohania duorara,Agmasoma penaei, and Pleistophora sp., microsporidian parasites of pink shrimp, Penaeus duorarum

The pathology of pink shrimp, Penaeus duorarum, infected with the microsporidians Thelohania duorara, Agmasoma penaei, and Pleistophora sp. was described. Infections of T. duorara were widespread in most tissues; spores were located throughout the hemocoel, at the periphery of all striated muscle bundles, and in muscle and connective tissue surrounding the digestive tract. A. penaei infections invaded only dorsal abdominal muscles, muscles adjacent to blood vessels, and ovaries. Infected muscles and ovaries were eventually completely destroyed. Masses of A. penaei spores were often engulfed by hemocytes. Pleistophora sp. infected the interior of all striated muscles. Infected muscles were never completely destroyed but were often atrophied.     

Black mat syndrome,an invasive mycotic disease of the tanner crab, Chionoecetes bairdi

Black mat syndrome, caused by an encrusting fungus on the exterior of the carapace of tanner crabs, has been known for many years. Although it is a nuisance when processing crabs for the commercial market, it has been thought to be restricted to the external surfaces of the crab and, therefore, nonpathogenic. In the present study, 11 tanner crabs from the Kodiak area of Alaska with and 9 without grossly recognizable masses of the fungus on the carapace were necropsied and examined histologically utilizing special stains selective for fungi. In all individuals with the syndrome, hyphae of the fungus, previously identified as Phoma fimeti, penetrated the carapace and virtually replaced the underlying epidermis. In the more advanced cases, the eyestalk was invaded and the epidermis destroyed, and hyphae extended into the eyestalk musculature and nervous tissue. To date, infections of the connective tissue sheaths surrounding the esophagus, stomach, heart, hemopoetic tissue, thoracic ganglion, antennal gland, and ovary have also been observed. None of the crabs without the syndrome contained internal hyphae. Although data on the lethality of the disease are not yet available, the ease with which the hyphae penetrate the chitinous exoskeleton, their extensive proliferation in the epidermis, and their ability to invade deep tissues causing obvious pathological effects, are highly suggestive that it is a virulent, probably fatal, disease that may have a significant impact on tanner crab population dynamics.     

Refuge utilization and preferences between competing intertidal crab species

Many invertebrates avoid predation risk by seeking and defending refuges that can be in limited supply, producing strong intra- and inter-specific interference competition. Previous experimental studies in central Chile demonstrated that interference competition for refuges is the primary factor driving habitat segregation between the predatory crabs Acanthocyclus gayi and A. hassleri, with the latter species monopolizing galleries inside mussel beds in the mid intertidal zone and limiting A. gayi to rock crevices. Yet, habitat partitioning between rival species can result from differences in habitat preferences and not solely from interference interactions. Moreover, since A. gayi is also known to shelter in turf-forming algae (predominantly Gelidium), which dominates extensive areas in the low intertidal zone, among-sites variation in the turf morphology and abundance could modify habitat preferences and the pattern of inter-specific interactions. We experimentally evaluated refuge habitat preferences of individual, similarly-sized adult A. gayi and A. hassleri in the laboratory, comparing choice patterns across multiple trials with paired combinations of the main refuge types commonly used by crabs in the field: a) mussel galleries, b) rock crevices, c) short algal turf, and d) tall algal turf. Our results showed that both species display a strong ranking of preferences for some refuge habitats over others. In general, mussel galleries were the preferred refuge type for both crab species, but their preference rankings changed depending on turf morphology. When turf was short, A. hassleri and A. gayi made identical refuge choices, strongly preferring mussel galleries over crevices and these over the short turf. In contrast, when the turf was tall A. gayi selected equally the tall turf or mussel galleries, and these were strongly preferred over crevices. A. hassleri, on the other hand, largely ignored tall turf and kept the highest preference for mussel galleries. A field experiment in which crabs were offered to foraging birds demonstrated that A. hassleri is more susceptible than A. gayi to predation by kelp gulls when outside refuges. Differences in patterns of coloration between crabs may underlie between-species differences in predation susceptibility and their habitat choices. These results suggest that the among-site differences in turf morphology (height and shape of fronds), which is largely driven by varying intensity of upwelling, could affect crab preferences for refuge habitats and the relative importance of inter-specific interference competition.     

Mangrove sesarmid crab feeding experiments in Peninsular Malaysia

The feeding ecology of mangrove sesarmid crabs in Peninsular Malaysia was investigated by field and laboratory experiments using four mangrove leaf species (Avicennia officinalis, Bruguiera gymnorrhiza, B. parviflora and Rhizophora apiculata) and leaves of different condition (fresh and senescent). Leaves tethered on strings at high (Bruguiera zone) and low (Rhizophora zone) intertidal positions, both upstream (Sungai Pasir) and downstream (Lower Merbok) showed significant amounts of leaf litter removal in 24 h (mean 79±3% initial dry mass). Significantly more B. gymnorrhiza was consumed in Bruguiera zones and significantly less senescent A. officinalis in the upstream Rhizophora zone. In Bruguiera zones, significant numbers of leaves were taken down burrows but there were no preferences for leaf species or condition of leaf taken down burrows at all sites. In 24 h, under laboratory conditions, the sesarmid crabs Sesarma (Perisesarma) eumolpe and S. (Perisesarma) onychophorum were offered with a mangrove species choice of either fresh or senescent leaves. There was no difference in mangrove species taken when the leaves were senescent for both crab species, but when the leaves were fresh, significantly more A. officinalis leaves were consumed by both sesarmid crab species. S. onychophorum ate significantly more B. parviflora than did S. eumolpe. The crab distribution in the field was related to the preferred tree species dominance, indicating that tree species may be important for crab species distribution, or vice versa. The mean rate of leaf consumption was not significantly different between the crab species; S. eumolpe was 29.9±5.9 and S. onychophorum was 35.3±7.2 mg dry mass per wet mass gram of crab in 24 h. Rhizophora spp. were the least preferred species in all feeding experiments, a finding which may have implications for ecosystem functioning in monoculture rehabilitation projects.     

Heart and scaphognathite activity in the shore crab Carcinus maenas (L.)

Simultaneous recordings of the activity of the heart and both scaphognathites of Carcinus maenas (L.) were investigated during a wide repertoire of normal activities. The scope of activity for the heart and scaphognathites could usefully be subdivided into three smaller ranges or levels which, although arbitrary, were recognizable on the basis of frequency and arrhythmic phenomena.Alterations of heart and scaphognathite activity induced by a variety of laboratory treatments were investigated in order to evaluate and aid the interpretation of such data for comparative purposes. Providing the recent manipulative history of the animal is known, the behaviours of heart and scaphognathites have been shown to be predictable and useful as sensitive sources of comparative data.The observed behaviour of the heart and scaphognathites has been discussed in terms of their nervous control systems and of their implications in a variable system of blood circulation and gill ventilation.     

Potassium retention in crab nerve

Spectrophotometric determinations of the potassium content of small volumes of sea water brought into contact with crab nerves over half-hour intervals demonstrate (a) a continuous loss of potassium from the fibers while at rest in oxygen; (b) an appreciable increase in the leakage during anoxia; (c) a reabsorption of potassium during postanoxic recovery; and (d) a reduction in the amplitude of the anoxic and postanoxic changes in the presence of glucose.     

The composition of the blood of the grapsid crab, Helice crassa Dana

The ability of the grapsid crab, Helice crassa Dana, to regulate the ionic concentration of its blood in adverse salinities has been investigated. The significance of these data is compared with those for other grapsid crabs and other Crustacea.     

Heat-labile Enzymes in a Psychrophilic Bacterium

The oxidative and fermentative activities of a psychrophilic bacterium (strain 82), whose maximal growth temperature is 35 C, were completely destroyed by exposure of the cells to 46 C for about 1 hr, whereas those of mesophilic Escherichia coli were unaffected. Similar results were obtained with cell-free extracts. In attempts to determine some of the specific enzymes inactivated in strain 82 by exposure to 46 C, it was found that reduced nicotinamide adenine dinucleotide oxidase was completely inactivated at 46 C in 2 hr. Also, cytochrome c reductase was completely destroyed at 46 C in 1 hr and was 70% destroyed at 40 C in 2 hr. The heat lability of the latter may determine the maximal growth temperature of the organism. In addition, the results indicated that the enzymes of strain 82 involved in the clastic split of pyruvate to formate and acetate are inactivated by exposure to 46 C and that the lactic and glycerol dehydrogenases are more heat-labile than those in E. coli. Succinic, nicotinamide adenine dinucleotide phosphate-alcohol, and glucose-6-phosphate dehydrogenases, however, in both strain 82 and E. coli, were essentially unaffected by exposure to 46 C for 2 hr.     

Hermit crab biocoenoses: a worldwide review of the diversity and natural history of hermit crab associates

The symbiotic associates of hermit crabs (excluding parasites and flora) are reviewed worldwide. The review includes species found on the shells occupied by hermit crabs (epibiotic species), species boring into these shells (endolithic species), species living within the lumen of the shell (either free-living or attached to the shell), species attached to the hermit crabs themselves, and hypersymbionts. In total over 550 invertebrates, from 16 phyla are found associated with over 180 species of hermit crabs. Among these associates, 114 appear to be obligate commensals of hermit crabs, 215 are facultative commensals, and 232 are incidental associates. The taxa exhibiting the highest number of associates are arthropods (126), polychaetes (105), and cnidarians (100). The communities of species associated with Dardanus arrosor, Paguristes eremita, Pagurus bernhardus, Pagurus cuanensis, and Pagurus longicarpus are the best studied and harbor the most diverse assemblages of species. While trends in biodiversity of hermit crab assemblages do not follow predicted patterns (e.g., hermit crabs within the Indo-West Pacific do not harbor more species than those from temperate regions), this is suggested to reflect a lack of sampling rather than a true representation of the number of associates. Hermit crabs date to at least the Cretaceous and provided a niche for a number of groups (e.g., hydractinians, bryozoans, polydorids), which were already associates of living gastropods. Apparently hermit crab shells initially supplied a substrate for settlement and then these symbiotic relationships were reinforced by enhanced feeding of symbionts through the activity of the hosts. Through their use and recycling of gastropods shells, hermit crabs are important allogenic ecosystem engineers in marine habitats from the intertidal to the deep sea. Hermit crabs benefit from some symbionts, particularly cnidarians and bryozoans, through extension of shell apertures (alleviating need to switch into new shells) and by providing protection from predators. However, hermit crabs are also negatively impacted (e.g., decreased reproductive success, increased predation) by some symbionts and a review of egg predators is provided. Thus, the symbiotic relationships between hermit crabs and many associates are difficult to characterize and often exhibit temporal changes depending on environmental and biological factors. Research on the biology of these symbionts and the costs/benefits of their associations with hermit crabs are analyzed. While some associates (e.g., Hydractinia spp.) have been studied in considerable detail, for most associations little is known in terms of the impacts of symbionts on hosts, and future experimental studies on the multitude of relationships are suggested.     

Offspring recognition by mother crab spiders with extreme maternal care

Maternal care is provided by several spider species, but there are no reports of mother spiders recognizing their young, which suggests that maternal care can be exploited by unrelated individuals. Diaea ergandros, a crab spider with extreme, sacrificial maternal care, does accept unrelated spiderlings (ca. 43.9% of spiderlings) into its nest in areas of high nest density. However, a field and a laboratory experiment with mother spiders and natural and adoptive spiderlings demonstrated that mothers did recognize their own offspring. Recognition was not expressed in survival as adopted (unrelated) spiderlings had similar survival rate to that of natural offspring. Instead it was displayed in growth; mother D. ergandros caught large prey items for their own offspring, but not for adopted spiderlings, and so natural offspring grew more than adopted spiderlings. Also, mothers produced trophic oocytes, which are important for the sacrificial care that influences spiderling survival, only when they lived with their own offspring.     

Antimicrobial defense mechanisms in the horseshoe crab,Limulus polyphemus: Effect of sodium chloride on bactericidal activity

Bactericidal activity of heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) was found to be inhibited by low molar (10 to 100 mm) concentrations of the monovalent cations Na⁺ and K⁺. The protective effect of Na⁺ was shown to be species dependent and inversely proportional to the sensitivity of the test microorganism as determined by bactericidal titer. The effect of Na⁺ (100 mm) on bactericidal activity in whole (unheated) LAL, when assayed against a sensitive species (Escherichia coli), was negligible and in direct contrast to data obtained when using 60°C extracts of LAL. Increasing the concentration of Na⁺ to reflect levels in L. polyphemus blood (400 to 500 mm) afforded only minimal protection for sensitive bacterial species. These data suggested that whole LAL may contain a heat-labile substance (protein?) capable of binding Na⁺, obviating the inhibitory effect of Na⁺ on bactericidal activity. Further evidence for the existence of this cation-binding substance was obtained when 55°C extracts of LAL were found refractory to Na⁺ concentrations which totally abolished bactericidal activity in 60°C extracts. The mechanism of Na⁺ inhibition of bactericidal activity and the role of the cation-binding substance in L. polyphemus host defense against bacterial invasion and dissemination is discussed.     

Antibacterial Activity of the Lactoperoxidase System in Milk Against Pseudomonads and Other Gram-Negative Bacteria

Products of thiocyanate oxidation by lactoperoxidase inhibit gram-positive bacteria that produce peroxide. We found these products to be bactericidal for such gram-negative bacteria as Pseudomonas species and Escherichia coli, provided peroxide is supplied exogenously by glucose oxidase and glucose. By the use of immobilized glucose oxidase the bactericidal agent was shown to be dialyzable, destroyed by heat and counteracted, or destroyed by reducing agents. Because the system is active against a number of gram-negative bacteria isolated from milk, it may possibly be exploited to increase the keeping quality of raw milk.     

Development and validation of a sensitive radioimmunoassay for naturally occurring beta-endorphin-like peptides in human plasma

β-Endorphin-like peptides in blood plasma of normal human subjects were studied by means of a radioimmunoassay (RIA) and gel filtration. Plasma was extracted with silica gel, which was washed with water and 1 n HCl, and eluted with 50% acetone. Plasma extracts thus obtained and standard synthetic human β-endorphin yielded parallel RIA curves. Total immunoreactivity in normal donors ranged from 1.2 to 10.4 fmol/ml (21 subjects). The immunoreactivity was completely destroyed by treatment with papain. Gel filtration indicated the presence of three components-one of unknown nature at the void volume and the others at elution positions characteristic of β-lipotropin and β-endorphin. Recoveries of human β-endorphin and β-lipotropin added to plasma were 53 and 58%, respectively. Addition of N-ethylmaleimide to plasma or of aprotinin to blood immmediately following collection had no effect on the amount of total immunoreactivity. Furthermore, a large amount of β-endorphin-like immunoreactivity. The above results lead us to conclude that a β-endorphin-like immunoreactive peptide occurs naturally in plasma of normal human subjects.     

Ocean acidification impairs crab foraging behaviour

Anthropogenic elevation of atmospheric CO₂ is driving global-scale ocean acidification, which consequently influences calcification rates of many marine invertebrates and potentially alters their susceptibility to predation. Ocean acidification may also impair an organism''s ability to process environmental and biological cues. These counteracting impacts make it challenging to predict how acidification will alter species interactions and community structure. To examine effects of acidification on consumptive and behavioural interactions between mud crabs (Panopeus herbstii) and oysters (Crassostrea virginica), oysters were reared with and without caged crabs for 71 days at three pCO₂ levels. During subsequent predation trials, acidification reduced prey consumption, handling time and duration of unsuccessful predation attempt. These negative effects of ocean acidification on crab foraging behaviour more than offset any benefit to crabs resulting from a reduction in the net rate of oyster calcification. These findings reveal that efforts to evaluate how acidification will alter marine food webs should include quantifying impacts on both calcification rates and animal behaviour.