The Circadian System of Crustaceans

Crustaceans exhibit a variety of overt circadian rhythms. Observations on intact animals suggest the existence of more than one circadian pacemaker in the nervous system. Ablation experiments so far have been inconclusive in pinpointing the location of putative pacemakers. However, various structures, most notably the optic peduncle, have been shown to sustain circadian rhythmicity in vitro. Retinal sensitivity and neurosecretory activity display circadian rhythms in the isolated optic peduncle, but they are also responsive to synchronizing influences from other regions of the central nervous system, most notably the supra-esophageal ganglion. A model based on a number of circadian pacemakers distributed in the central nervous system best fits the experimental results at present. Coupling of rhythmicity between independent circadian pacemakers is likely to occur, and a neuroendocrine stage of integration has been proposed for several rhythms. Various entraining agents have been identified, and more than one may play a part in the synchronization of a given rhythm.     

Role of Pore Size Location in Determining Bacterial Activity during Predation by Protozoa in Soil

The predation of a luminescence-marked strain of Pseudomonas fluorescens by the soil ciliate Colpoda steinii was studied in soil microcosms. Bacterial cells were introduced in either small (neck diameter, <6 (mu)m) or intermediate-sized (neck diameter, 6 to 30 (mu)m) pores in the soil by inoculation at appropriate matric potentials, and ciliates were introduced into large pores (neck diameter, 30 to 60 (mu)m). Viable cell concentrations of bacteria introduced into intermediate-sized pores decreased at a greater rate than those in small pores, with reductions in bacterial populations being accompanied by an increase in viable cell numbers of the ciliate. The data indicate that the location of bacteria in small pores provides significant protection from predation. In the absence of C. steinii, the level of metabolic activity of the bacterial population, measured by luminometry, decreased at a greater rate than cell number, and the level of luminescence cell(sup-1) consequently decreased. The decrease in levels of luminescence indicates a loss of activity due to starvation. During predation by C. steinii, the level of the activity of cells introduced into small pores fell in a similar manner. The level of cell activity was, however, significantly greater for cells introduced into intermediate-sized pores, despite their greater susceptibility to predation. The data suggest that increased activity arises from a release of nutrients by the predator and the greater accessibility of bacteria to nutrients in larger pores. Nutrient amendment of microcosms resulted in increases in bacterial populations to sustained, higher levels, while levels of luminescence increased transiently. The predation of cells introduced into intermediate-sized pores was greater, and there was also evidence that the level of activity of surviving bacteria was greater for bacteria in intermediate-sized but not small pores.     

Spontaneous Activity of Murine Neuromuscular Junctions in the Presence of Dantrolene

By recording miniature end-plate potentials (mEPP), the effects of dantrolene (10-100 M), a blocker of ryanodine receptors, were studied on the isolated diaphragm of mice. The effects to be studied were as follows: on spontaneous secretion of acetylcholine quanta and on the pattern of interaction with ryanodine effects. Two-hour-long application of dantrolene to the muscle caused no significant changes in the amplitude and dispersion (²) of mEPP, nor on its time course. In the presence of 100 M dantrolene, the mean frequency of mEPP increased, on average, by 58.3 ± 5.9% (P < 0.05). Dantrolene suppressed in a dose-dependent manner a number of ryanodine effects (this agent was used in a concentration of 0.5 M as an intensifier of intracellular Ca²⁺ mobilization): it completely prevented the appearance of the population of high-amplitude (the so-called giant) mEPP, reduced by 50-80% the increment of the mEPP amplitude dispersion, and increased by 25-45% the mEPP mean amplitude; the above effects were induced by ryanodine application for 120 min. After preliminary application of dantrolene (10-100 M), ryanodine caused an effect not observable in the absence of dantrolene: mEPP became more frequent (140-210%). Thus, when acting on motor synapses, dantrolene behaves as a nontoxic agent, inducing only a presynaptic effect – a moderate increase in the mEPP frequency. The dual character of interaction between dantrolene and ryanodine in motor synapses was observed: on the one hand, dantrolene acts as a physiological antagonist of ryanodine by reducing the ryanodine-induced increase of dispersion and mEPP amplitude; on the other hand, dantrolene unmasks the ability of ryanodine to increase the mEPP frequency.     

Recognition of Non-self in Crustaceans

This paper reviews the current concepts of recognition of non-selfin crustaceans and relates these concepts to recognition inthe invertebrates in general. It focuses primarily on a decapodcrustacean, the blue crab(callinectes sapidus)and on resultsusing this animal as a model to study the clearance of virusesand xenogeneic proteins. Clearance studies indicate that bluecrabs possess a quasi-specific recognition system in the normalor "non-immunized"state. This system is capable of rapidly clearingforeign proteins and certain viruses from the circulation, andresults in concentration of such proteins into the gills andviruses into the hepatopancreas or gills. Although humoral factorswhich bind foreign proteins or neutralize viruses have beenisolated, transfusion exchange experiments involving depletionof circulating cells and/or humoral factors indicate that theblue crab does not require circulating hemocytes or humoralfactors for clearance of foreign proteins. These results suggestthat a population of fixed cells, possibly those in the gills,may be the critical component for recognition of foreign proteinsby normal crabs.     

Cell-Cell Recognition in the Regenerating Neuromuscular System of the Cockroach

SYNOPSIS. The neuromuscular system of the cockroach containsmotor neurons and muscles that can be identified in all individualinsects When the axons of these motor neurons are damaged theyregenerate and eventually reform synapses only with the originaltarget muscles However at early times after axotomy transientinappropriate functional connections are made between regeneratingneurons and muscles that theynever normally innervate Laterthe inappropriate synapses are inactivated, the inappropriateaxon branches eliminated and the original innervation patternreformed A cellcell recognition between identified motor neuronsand muscles is required to explain these observations, particularlyin light of experiments demonstrating the absence of competitionbetween appropriate and inappropriate axon terminals withinthe muscle. A minimum biochemical requirement of such a cell-cell recognitionis the existence of molecules whose presence in muscles correlateswith the innervation by identified motor neurons Using fluoresceinlabelled plant lectins to detect muscle surface glycoproteinssuch molecules have been identified In addition, there shouldbe molecular differences among the surfaces of the axon terminalsof the various identified motor neurons Hybrid oma techniqueshave enabled us to obtain monoclonal antibodies that bind tosurfaces of axon terminals of some motor neurons and not othersThese lectin receptors and antigens are good candidate recognitionmacromolecules Other molecules essential for axonal regenerationhave been identified by their presence in embryonic and adultregenerating neurons and their absence from intact adult neurons.     

The Phenomenon of Refusal in Muscular Activity. The Role of the Respiratory System

Among the factors leading to the refusal to continue muscular work, afferent impulsation carrying information about the efforts developed by the working muscles and about the respiratory function tension is noteworthy. Based on this information, the integral self-rating of the intensity of the physical load and its endurance is formed in the sensory CNS sphere, with one of the signals to stop the work being the feeling of shortness of breath. Under the cyclic working conditions, the so-called critical power load, which is high enough but does not lead to a refusal so far, enabling one to perform the maximum amount of useful work, appears to be the most effective. A question is posed as to the possibility of using an active choice of preferable load intensity by an individual in order to develop a method for determining the optimal work power according to the functional state of the body.     

To the Question of Eyes in Primitive Crustaceans

Based on a review of studies of functional and comparative anatomy of crustacean eyes, a hypothesis is formed on eyes in primitive crustaceans. It is suggested that they were similar to present-day frontal eyes of the malacostracan type. Neuronal architecture suggests that the development to apposition compound eye followed two main routes, one seen in malacostracans and the other in non-malacostracans. Within the two subgroups different and separate lines have been followed to form specialized apposition types and, within the Malacostraca, superposition eyes as pointed out by Nilsson (Nilsson, D. E. 1989. Facets of vision, pp. 30–73).     

Role of Plant Residues in Determining Temporal Patterns of the Activity,Size, and Structure of Nitrate Reducer Communities in Soil

The incorporation of plant residues into soil not only represents an opportunity to limit soil organic matter depletion resulting from cultivation but also provides a valuable source of nutrients such as nitrogen. However, the consequences of plant residue addition on soil microbial communities involved in biochemical cycles other than the carbon cycle are poorly understood. In this study, we investigated the responses of one N-cycling microbial community, the nitrate reducers, to wheat, rape, and alfalfa residues for 11 months after incorporation into soil in a field experiment. A 20- to 27-fold increase in potential nitrate reduction activity was observed for residue-amended plots compared to the nonamended plots during the first week. This stimulating effect of residues on the activity of the nitrate-reducing community rapidly decreased but remained significant over 11 months. During this period, our results suggest that the potential nitrate reduction activity was regulated by both carbon availability and temperature. The presence of residues also had a significant effect on the abundance of nitrate reducers estimated by quantitative PCR of the narG and napA genes, encoding the membrane-bound and periplasmic nitrate reductases, respectively. In contrast, the incorporation of the plant residues into soil had little impact on the structure of the narG and napA nitrate-reducing community determined by PCR-restriction fragment length polymorphism (RFLP) fingerprinting. Overall, our results revealed that the addition of plant residues can lead to important long-term changes in the activity and size of a microbial community involved in N cycling but with limited effects of the type of plant residue itself.Modern agricultural practices include a return of plant residues to soil, as this is considered sustainable to the environment. It is now recognized that the conversion of native land into cultivated systems leads to carbon losses, which can be up to 20 to 40% (17). Postharvest plant residues therefore represent an important source of carbon, helping to replenish soil organic matter that decomposes as a result of cultivation. Decomposing plant residues are also a source of nutrients, such as nitrogen, with reduced nitrate leaching compared to mineral fertilizers, which is beneficial for water quality (3). In addition, leaving the plant residue on the soil surface limits water losses by evaporation and prevents soil erosion by wind or water (15).The biochemical composition of plant residues is one of the most important factors influencing their decomposition in soil (14, 28, 29, 51). Indeed, Manzoni et al. (28), using a data set of 2,800 observations, showed previously that the patterns of decomposition were regulated by the initial residue stoichiometry. Several other factors such as climatic conditions, soil type, or localization of the residue in the soil (incorporated or on the soil surface) were also reported previously to influence decomposition (2, 24, 29, 44). Microorganisms are the major decomposers of organic matter in soil, and therefore, the diversity and activity of the microbial community during plant residue decomposition has received much attention (6, 23, 26, 27, 35). It was shown previously that the biochemical composition of plant residues influences microbial respiration (8) and microbial community structure (7, 37). The recent development of carbon-labeling approaches has furthered our knowledge of the microorganisms that actively assimilate the carbon derived from various plant residues (10, 31). However, most of those studies focused on microorganisms involved in C mineralization, and in contrast, very little is known about the effect of plant residue decomposition on the microbial communities involved in biochemical cycles other than the carbon cycle. Thus, despite the influence of plant residues on nitrogen cycling (1, 4, 5, 16, 20), studies assessing the effect of the presence and composition of plant residues on the ecology of microbial communities involved in nitrogen cycling are rare (21, 32, 36).The dissimilatory reduction of nitrate into nitrite is the first step in the processes of denitrification and the dissimilatory reduction of nitrate to ammonium (33, 41). The reduction of nitrate by denitrification leads to losses of nitrogen, which is often a limiting nutrient for plant growth in agriculture. Two types of dissimilatory nitrate reductases, differing in location, have been characterized: a membrane-bound nitrate reductase (Nar) and a periplasmic nitrate reductase (Nap) (9, 53). Nitrate reducers can harbor either Nar, Nap, or both (40, 47). Nitrate reducers are probably the most taxonomically diverse functional community within the nitrogen cycle, with members in most bacterial phyla and also archaea (42). Because of this high level of diversity of heterotrophs sharing the ability to produce energy from nitrate reduction, nitrate reducers are an excellent model system to investigate the response of the N-cycling community to plant residue addition.The aim of this work was to determine how the incorporation of plant residues with contrasting biochemical compositions into soil affects the nitrate-reducing community. For this purpose, we monitored the dynamics of the potential activity, size, and structure of the nitrate-reducing community after the addition of wheat, rape, or alfalfa residues to soil in a field experiment. As the nature and availability of the substrate change during residue decomposition (38, 39, 48), the influence of the incorporation of different plant residues on the nitrate-reducing community was investigated at several sampling times for 11 months.     

Occurrence of Hemocyanin in Ostracod Crustaceans

Hemocyanin is a copper-containing protein that transports O₂ in the hemolymph of many arthropod species. Within the crustaceans, hemocyanin appeared to be restricted to Malacostraca but has recently been identified in Remipedia. Here, we report the occurrence of hemocyanin in ostracods, indicating that this respiratory protein is more widespread within crustaceans than previously thought. By analyses of expressed sequence tags and by RT-PCR, we obtained four full length and nine partial hemocyanin sequences from six of ten investigated ostracod species. Hemocyanin was identified in Myodocopida (Actinoseta jonesi, Cypridininae sp., Euphilomedes morini, Skogsbergia lerneri, Vargula tsujii) and Platycopida (Cytherelloidea californica) but not in Podocopida. We found no evidence for the presence of hemoglobin in any of these ostracod species. Like in other arthropods, we identified multiple hemocyanin subunits (up to six) to occur in a single ostracod species. Bayesian phylogenetic analyses showed that ostracod hemocyanin subunit diversity evolved independently from that of other crustaceans. Ostracod hemocyanin subunits were found paraphyletic, with myodocopid and platycopid subunits forming distinct clades within those of the crustaceans. This pattern suggests that ostracod hemocyanins originated from distinct subunits in the pancrustacean stemline.     

Aspects of Lipid Metabolism in Crustaceans

Lipid is the predominant organic reserve of many crustaceansand is important in the metabolism of many of these animals.Ingested lipid is digested by gastric lipase and apparentlyabsorbed into depot-lipid as rß-monoglycerides. Thevariation in the content and composition of the depot-lipidis a function of both the external environment and internalcontrol systems. Evidence suggests that lipids from marine organismscontain more long-chain polyunsaturated fatty acids than doesthe lipid of fresh water organisms which in turn have a highproportion of C₁₆ and C₁₈ fatty acids. The fatty-acid compositionof the sub-tropical land crab,Gecarcinns lateralis, resemblesthat of the fresh-water crustaceans. In addition, our studiesindicate that aspects of lipid metabolism may be under endocrinecontrol. The induction of premolt by destalking markedly increasesthe synthesis of lipid from metabolic precursors and its subsequentincorporation into the depot-lipid of the hepatopancreas. Inthe late premolt stages there is a decrease in the lipid contentof the hepatopancreas. This occurs as the lipid is mobilizedfrom the hepatopancreas to meet the energy demands of all thoseprocesses resulting in ecdysis. This sinusoidal variation inthe lipid metabolism of the hepatopancreas is influenced byan eyestalk factor (s).     

Ontogeny of Vision in Marine Crustaceans

Marine crustaceans present an extremely interesting set of examplesin which to examine visual development and metamorphosis. Larvaeof these animals are almost always planktonic, living in thelight field of open waters. The presence of a simple, predictablephotic environment, the relatively basic visual requirementsof larvae, and the need to remain transparent to reduce predationlead to the use of a single eye type throughout all marine crustaceanlarvae. Adult crustaceans, on the other hand, use a greaterdiversity of optical designs than all other animals combined,occupy habitats from the deep sea to mountaintops, and havevery complex visual systems and behaviors. Thus, visual developmentvaries tremendously among modern Crustacea. In this brief review,we consider the structure and development of marine crustaceaneyes, focusing on optics, retinal design, and metamorphosisof the visual pigments.     

Hormones in the Lives of Crustaceans: An Overview

We present an overview of the isolation and characterizationof three hormones (or hormone families) important for the growthand development of decapod crustaceans. These hormones includethe ecdysteroids (steroid molting hormones), the hyperglycemichormone neuropeptide family, and the terpenoid methyl farnesoate.Using examples primarily from our laboratory, we describe workon these hormones using various life stages of the lobster (Homarusamericanus) as the principal model.     

Acetylcholine Receptor Binding Properties at the Rat Neuromuscular Junction During Aging

Specific binding characteristics of acetylcholine receptors at the diaphragm neuromuscular junction of rats aged 10 (mature adult) and 28 (aged) months were assayed by measuring 125I-alpha-bungarotoxin binding. Maximal binding to intact tissue samples was greater in the older rats; this could be attributed to an age-related increase in terminal branching. The toxin concentration at which half-maximal binding occurred increased in the older rats. Binding kinetics were assayed in finely minced tissue samples, and the association rate constant was observed to decrease in the 28-month animals. Retardation of the initial rate of toxin binding by d-tubocurarine (dTC) in minced tissue was described by a two-component nonlinear Hofstee plot; IC50 values (7.1-7.2 microM and 39.0-46.5 nM) were about the same for both age groups, but there was a significant shift toward the low-affinity values in the aged rats. Rhodamine-conjugated alpha-bungarotoxin was used to visualize receptor localization. There were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. The data indicate a shift toward lower binding affinity during aging, which may involve changes either in one of the two toxin-binding sites on individual receptors, in dTC blocking of the channel moiety, or in receptor types.     

Neuroendocrine Correlates of Circadian Rhythmicity in Crustaceans

The secretion of neurohormones from the crustacean X-organ –sinus gland system is controlled by environmental influences,light being the most conspicuous. Two sets of photoreceptorsappear to mediate the influence of light on neurosecretion basedon intracellular recordings from X-organ neurons and estimationsof hormone release. Extra-retinal photoreceptors can initiateneurohormonal release from the eyestalk. Neurosecretory activity is also influenced by putative neurotransmitters.GABA is found in high concentrations in the medulla temninalisof the eyestalk and is released by stimulation, in a calcium-dependentmanner. Diurnal variations occur in the amounts of eyestalk neurohormones,either those present in the eyestalk or released by electricalstimulation of the isolated sinus gland. Rhythm phases varyfrom one hormone to another. Neurohormones secreted in the eyestalkare also found in other regions of the central nervous system.Rhythms of neurosecretion are present both in the secretionin the isolated eyestalk and in eyestalkless animals, thus indicatingthat rhythmicity is a distributed property of the neurosecretorysystem.     

Crustaceans and rotifers in the predatory feeding of Utricularia

The contents of bladders in the predatory plant Utricularia from three waterbodies were studied. The composition and number of prey depended on the development of zooplankton and phytophilous fauna in the environment. The probability of getting the prey into the bladder was determined by the specificity of the prey’s behavior and size. The most numerous prey were predatory Copepoda and nonpredatory Cladocera, which use the bladderwort as a substrate. The shares of other cladocerans in the bladders were inconsiderable when there were high densities of these species in the waterbody. No negative impact of the bladderwort on the rotifer abundance was revealed.     

A Method for Determining the Activity State of Hair Follicles

A histological method is described for determining the proportion of growing hair follicles h skin samples. A variation of the Sacpic staining method, modified for bulk processing, produces high contrast staining of the principal tissue types present in skin. In particular, the inner root sheath is accentuated, facilitating detection of active follicles. Skin preparations from a range of species are used to illustrate structural characteristics of follicles viewed in cross section at various stages of the hair cycle and to establish criteria for classification of the state of activity of follicles. The hair cycle may be divided into quiescent and active states at the points of rapid transition (early pronanagen and mid catagen). Data from repeated skin biopsies from ferrets and goats are also used to demonstrate quantitative estimation of follicle activity, change in compound follicle size, and the relationship between follicle type and fiber medullation.