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Identification of a Juvenile Hormone-Like Compound in a Crustacean THE JUVENILE HORMONES (JHs) AREa family of sesquiterpenoid compounds that regulate both metamorphosis and gametogenesis in insects (1). With the discovery [...]

Burgess Shale Faunas and the Cambrian Explosion THE FOSSIL RECORD MAY BE THE BOOK OF ORGANIC HISTORY, but like the worst of chroniclers, the scribe responsible suffers from shocking lapses [...]

Parhyale hawaiensis, an amphipod crustacean, displays total (holoblastic) cleavage during early embryogenesis, and we hope to determine how tightly cell lineage and cell fates are coupled in this species. In Parhyale, the first two divisions are radial, total, and slightly asymmetrical. The third division is highly asymmetric and produces an embryo that consists of four macromeres and four micromeres. Lineage studies performed at the eight-cell stage demonstrate that each cell normally contributes to only one of each of the germ layers (Gerberding et al, 2002). However, it is not known if these cells are specified or determined at this early stage. Cell ablations are a classical method for addressing how lineage is coupled to cell fate, and utilizing this approach at the light cell stage, we find that there is much regulation which occurs between lineage restricted equivalency groups that give rise to the germline, mesoderm, and ectoderm. In addition, the molecular mechanisms involved in establishing the cell fate pattern in Parhyale are unknown. Therefore, we have taken a candidate gene approach to address this question. Armadillo/[beta]-catenin is one such gene, as it is known to be 56 involved in setting up cell fates in different organisms in a variety of cellular contexts. We have cloned the Parhyale ortholog of armadillo/[beta]-catenin and examined its expression by in situ hybridization. Interestingly, at the earliest stages of development, maternally provided armadillo is present in what may be analogous to the germplasm or nuage, that then becomes restricted specifically to the germline. Additional ablation experiments, blastomere isolations, and mis-expression/knockdown experiments should further reveal the mechanisms behind asymmetric cell division and cell fates in Parhyale.

Specialization of crustacean trunk appendages may require the presence and absence of certain hox genes. Hox genes are highly conserved transcription factors that play a role in regionalization of animal body plans. Previous comparative analysis of one hox gene, Ultrabithorax (Ubx), in a variety of crustaceans illustrated a correlation between boundaries of gene expression and limb specialization in the crustacean trunk. While most trunk segments express Ubx and develop walking legs, trunk segments lacking Ubx expression develop as feeding appendages called maxillipeds. We have analyzed the expression patterns of all hox genes present in the amphipod crustacean Parhyale hawaiensis. From this analysis, we propose that it is the combination of Sex combs reduced (Scr) and Antennapedia (Antp) expression, in the absence of Ubx expression, in trunk segments that controls maxilliped specification in Parhyale. We are testing this hypothesis using gene knockdown techniques to further understand the function of each gene during Parhyale development. Traditional RNAi methods of injecting double-stranded RNA into embryos does not produce a permanent reduction in gene expression nor phenotype for individual hox genes. We are we are currently designing an alternative method to continuously express inverted repeat transcripts throughout development to knockdown gene expression. These experiments may potentially highlight mechanisms by which changes in gene expression can influence the evolution and development of morphological diversity in crustacean appendages.

Copper is an essential element and acts as a cofactor for a number of key proteins, however, in excess, it is toxic. Na+,K+-ATPase is present in the ion-transporting type branchial filaments (chloride cells) of Astacus leptodactylus. Antibodies to Na+,K+-ATPase have been widely to identify chloride cells in fish and crustacean. Juvenile A. leptodactylus were exposed to waterborne copper (3 [micro]mol l-1) for 10 days and the branchial chloride cells were identified by Na+,K+-ATPase immunocytochemistry. The mean optical density of Na+,K+-ATPase stained chloride cells in branchial filaments epithelium of controls and copper exposure was 0.956[+ or -]0.024 (N=12) and 0.542[+ or -]0.019 (N=10), respectively. The number of stained chloride cells decreased from 89% in controls to 53% in copperexposed Astacus. We observed that copper affected the branchial cells: hemocyte accumulate in vessels and filaments, the epipodite is hypertrophied with a disorganized epithelium and hemolymphatic edema. In conclusion, copper exposure increases levels of necrosis and apoptosis of branchial chloride cells and necrotic and apoptotic cells have much lower Na+,K+-ATPase antigenicity than mature chloride cells.

Crustacean growth and development is regulated by the fundamental process, ecdysis. Ecdysteroids, the hormones responsible for this process, are produced in the Y-Organs (YO) and located in the cephalothorax, Synthesis of ecdysteroid is inhibited by molt-inhibiting hormone (MIH), a neurosecretory peptide produced in the X-Organ/Sinus Gland Complex, located in the eyestalks of the animal. Little is known about the components involved in this MIH signaling pathway, though recent research has confirmed the presence of Guanylyl Cyclase and Nitric Oxide Synthase in YO tissue. The roles of these two putative secondary messengers in this pathway will be evaluated using an in vitro assay technique. Tropical Land Crabs, Gecarcinus lateralis, were subjected to Eyestalk Ablation (ESA), 2 days prior to removal of the YOs. Removal of the eyestalks removes the source of MIH and subsequently stimulates ecdysteroid secretion by the YOs. Guanylyl Cyclase and Nitric Oxide Synthase stimulators/inhibitors were introduced to the YO culture and incubated for 4.5 hours. Determination on the effects these components had on ecdysteroid secretion has elucidated their roles in the MIH signaling pathway. Supported by NSF (IBN-0342982) and scholarship to AMG by the Arnold and Mabel Beckman Foundation.

Decapod crustaceans are well known as major predators in marine ecosystems but less attention is paid to their role as prey. Decapod chelae, as multi-functional appendages, record the influence of both predators and prey. Durophagous crabs have dimorphic chelae (heterochely) that are initially major right claws and minor left claws, however, non-fatal attacks that result in the removal of the right major claw will lead to a reversal in chelae dimorphism upon the next regenerative molt; a phenomenon known as reversed handedness. Since decapod chelae are common in modern and fossil assemblages, evidence of nonlethal injury from frequencies of reversed handedness may provide a proxy for predation intensity on decapods. The proportion of reversed handedness in a decapod population is used as a measure of predation intensity on the decapods. Because reversed handedness is the result of failed attacks, it is analogous to repair scar or incomplete drillholes frequencies commonly found in molluscan studies, and has been used similarly to make inferences about predation intensity on decapods. Here we test the utility of the reversed handedness metric using a probabilistic model that includes the injury rate and kill rate for each molt stage of a population of heterochelous decapods. The model simulates the proportion of reversed handedness observed in death assemblages; values that can subsequently be compared to the actual frequency of predation attempts on the population.

Paired Y-organs secrete ecdysteroid hormones that control cycles of growth and molting in crustaceans. Y-Organs are regulated, at least in part, by molt-inhibiting hormone (MIH), a peptide produced and released by the X-organ/sinus gland complex of the eyestalks. We are investigating cellular signaling pathways involved in regulation of Y-organ function. In the present studies, Y-organs were incubated in vitro in the presence of [sup.35]S-methionine and experimental agents that influence the cyclic AMP (cAMP) signaling pathway. In 4-hr incubations, 8-Br-cAMP (a cAMP analog) suppressed incorporation of [sup.35]S-met into Y-organ proteins; the effect was concentration-dependent. Addition of cholera toxin (a Gs protein activator), IBMX (a phosphodiesterase inhibitor), or forskolin (a adenylyl cyclase activator) likewise suppressed incorporation of [sup.35]S-met into Y-organ proteins. The effect of cholera toxin was antagonized by KT5720 (a protein kinase A inhibitor). Incorporation of [sup.35]S-met into Y-organ proteins was also suppressed by an extract of sinus glands. The combined results suggest that cAMP plays an important role in regulation of Y-organ function. We are currently investigating the link of protein synthesis to ecdysteroid production, and the possibility of cross-talk between cAMP and other cellular signaling pathways in Y-organs. Supported by the National Science Foundation (IBN-0213047).

While the ability of xeno-oestrogens, like 4-n-nonylphenol (4-NP), to disrupt the natural hormonal systems of aquatic vertebrates is well known, evidence for comparable endocrine disrupting effects in decapod crustaceans, particularly during their larval development, remains scarce. In this investigation, a polyclonal antiserum raised against an apolipovitellin purified from the mature ovaries of the intertidal prawn, Palaemon elegans, was found to be immunoreactive with a 86 kDa polypeptide (IRP) present in all the larval and early post-larval stages. An indirect ELISA technique, developed to quantify the levels of the immunoreactive proteins present in the soluble protein fraction of homogenates of P. elegans larvae, found the quantity of the IRP varied between 0.47-0.67 ng [micro] depending on stage. Exposure of P. elegans larvae to 4-NP at the environmentally realistic concentrations of 0.2-20 [micro]g [L.sup.-1], resulted in a significant, concentration independent, increase in IRP levels of 13-18%. Conversely, exposure to comparable concentrations of the natural oestrogen, 17a-oestradiol ([E.sub.=2]), significantly reduced IRP levels by up to 11% in a similar concentration independent manner. Significant fluctuations in IRP levels were correlated with exposure to 4-NP and [E.sub.2]. However, with the exception of a slight reduction in the survival of larvae exposed to 4-NP at 2 [micro]g [L.sup.-1], neither 4-NP nor [E.sub.2] had any significant effect on the development, growth or survival of P. elegans larvae.

Fenoxycarb and pyriproxyfen are insecticides that gain their toxicity by specifically acting as insect juvenile hormone agonists (JHA), and so are endocrine disruptors by design and effectively prevent larvae from maturing into adults. Efforts to assess the environmental effects of JHAs on nontarget populations of invertebrates have resulted in the utilization of several established estuarine crustacean models. Gravid female grass shrimp (Palaemonetes pugio) and mud crabs (Rhithropanopeus harrisii), species with different developmental plasticity, were collected and held at wet lab conditions (20 ppt salinity, 25 [degrees]C) until larval release. Larvae were collected

Comparative developmental and reproductive studies were performed on several species of estuarine crustaceans in response to three juvenile hormone agonists (JHAs). Larval development of the grass shrimp, Palaemonetes pugio, was greater than two orders of magnitude more sensitive to disruption by JHAs than was embryonic development. For 2 JHAs, developing larvae of the mud crab, Rhithropanopeus harrisii, exhibited reduced metamorphic success at lower concentrations than grass shrimp larvae. These responses suggest that the more rigidly controlled metamorphic process in crabs is more sensitive to compounds acting as endocrine disruptors than is the more plastic metamorphic pattern in shrimp. The final crab larval stage, the megalopa, was more sensitive to JHA exposure than earlier zoeal stages. Mud crab larvae exposed to fenoxycarb had reduced biomass and lipid content, particularly triglycerides and sterols. Concentrations of fenoxycarb which reduced the reproductive capacity in single life-cycle exposures of the estuarine mysid, Americamysis bahia, were similar to those concentrations which inhibited metamorphosis in grass shrimp. Juvenile mysids released by exposed adults and reared through maturation without further exposure produced fewer young and had altered sex ratios (lower percentages of males) at lower parental-exposure concentrations than directly impacted parental reproduction. Since the endocrine glands responsible for regulating mysid sexual differentiation and reproduction develop during larval stages, these transgenerational responses may well be a product of irreversible effects during developmental exposures which become apparent following maturation and initiation of reproduction.

We are examining mechanisms governing transmitter release efficacy at two vastly different crustacean motor neurons. Phasic nerve terminals release 100-1000 fold transmitter per action potential than tonic nerve terminals, although they share the same postsynaptic muscle cells. We hypothesized that the transmitter release mechanism of phasic neurons possesses a greater sensitivity to calcium, and thus produces a greater amount of tranmsitter release. We tested this using photoactivation of 'caged calcium' while recording transmitter release from individual boutons. We found that phasic terminals exhibit a lower calcium threshold at which release begins to occur, and release transmitter at a higher rate for a given calcium concentration. These preliminary results support our hypothesis that transmitter release of phasic neurons is more sensitive to calcium entry than that of tonic neurons.

The habitats of most aquatic crustaceans are highly variable, particularly with respect to changing levels of oxygen. Fluctuations in factors such as salinity, temperature, and productivity levels can have significant effects on the amount of oxygen available in the medium. Some estuarine species of crustaceans are not only subject to short term hypoxia as would be experienced due to a tidal cycle, but also to long term seasonal hypoxia. We are interested in investigating the possible role of HIF-1a in the ability of hypoxia tolerant crustaceans to adapt to fluctuations in oxygen availability via oxygen-dependent changes in gene expression. Some crustaceans have been shown to differentially regulate the subunits of their oxygen transport proteins in response to low oxygen in order to change the oxygen affinity of the molecule, but the regulatory mechanism in crustaceans has not yet been demonstrated. We have identified a HIF-1a homologue in the brachyuran crustacean Cancer magister that is highly similar to the HIF-1a homologue, SIMA, from Drosophila melanogaster. It appears that the HIF-1 system is present in crustaceans and probably plays a significant role in regulating gene expression in an oxygen dependent manner. Supported by an NSFIGERT Fellowship, Eugene and Clarissa Evonuk Graduate Fellowship, and NSF 9984202.

The Malacostraca is the most speciose and morphologically diverse class in the Crustacea, with approximately 30,000 described species. However, because of this diversity in body plan it has been proposed that morphological characters in malacostracan systematic studies may be confounded by parallel or convergent evolution. Molecular phylogenetic studies would be a way to both test possible morphological convergences and to add new lines of evidence to strengthen the phylogenetic hypotheses. Yet, to date, few molecular phylogenetic analyses have been done for this group. Some of the major questions about the relationships within the Malacostraca are: Is the Leptostraca the sister group to the rest of the Malacostraca? What is the relationship between the five major groups (Hoplocarida, Phyllocarida, Syncarida, Peracarida, and Eucarida) within the Malacostraca? Is the Peracarida monophyletic? Are the Amphipoda and Isopoda sister groups? We will present 18S and partial 28S rDNA data to help answer some of these questions. We collected and sequenced 60 species within the Malacostraca representing 29 families, with heavy taxon sampling in the Peracarida. Nine other crustacean species were used as outgroups. Alignments were done with ClustalX and POY, and tree building was done using both parsimony and likelihood as the optimality criterion. Among some of our preliminary results are the placement of mysids as a basal group outside of the Peracarida, a sister group relationship between the Amphipoda and Isopoda, and a clarification of the relationships within the Amphipoda.

Motor neurons of arthropods present combinations of physiological and morphological properties which endow them with unique performance abilities. Within genetically specified limits, adaptive modification of their properties can occur both during development and after maturation. Extreme differences in neuromuscular performance are found among phasic and tonic motor neurons, especially in crustaceans. Analysis of synaptic transmission has led to definition of differences in response properties of synaptic vesicle exocytosis and in releasable synaptic vesicles. The genetically tractable neuromuscular junctions of Drosophila afford opportunities for elucidating the key factors that create synaptic differences.

A study was conducted to analyze rhythmic patterns of abundance in small sublittoral crustaceans. The synchronization of the patterns with tidal cycles was also examined. Experimental results indicated that small crustaceans may respond to changes of hydrologic variables that decline with tides. Findings also showed that patterns may correlate with a weak diel vertical movement between day and night.

A study was conducted to analyze adaptive processes associated with reproduction in populations of the Northern krill, Meganyctiphanes norvegica. Moult stages and seasonal variations of the inter-moult period were examined. The ovarian structure and egg production pattern were then analyzed. Results indicated that temperature significantly influenced the growth and egg production during reproductive seasons.

Research was conducted to examine the acoustic discrimination of two zooplankton species using bifrequency sonar 38 kHz and 120 kHz. Two species of Mysidacea, which were similar in shape but not in size and with differing behavioral patterns, were observed and discriminated. Only Siriella jaltensis was detected at 38 kHz while both were observed at 120 kHz. Results indicate that the 38 kHz is a good frequency to detect Siriella while 120 kHz is not a good frequency for the detection of the larger Siriella.