Longitudinal tracking of hemocyte populations in vivo indicates lineage relationships and supports neural progenitor identity in adult neurogenesis.

Adult neurogenesis, which takes place in both vertebrate and invertebrate species, is the process by which new neurons are born and integrated into existing functional neural circuits, long after embryonic development. Most studies in mammals suggest that self-renewing stem cells are the source of the new neurons, although the extent of self-renewal is a matter of debate. In contrast, research in the crayfish Procambarus clarkii has demonstrated that the neural progenitors producing adult-born neurons are capable of both self-renewing and consuming (non-self-renewing) divisions. However, self-renewing divisions are relatively rare, and therefore the production of adult-born neurons depends heavily on progenitors that are not replenishing themselves. Because the small pool of neural progenitors in the neurogenic niche is never exhausted throughout the long lives of these animals, we hypothesized that there must also be an extrinsic source of these cells. It was subsequently demonstrated that the neural progenitors originate in hemocytes (blood cells) produced by the immune system that travel in the circulation before ultimately integrating into niches where the neural lineage begins. The current study examines the developmental lineage of the three hemocyte types - hyaline (HC), semigranular (SGC) and granular (GC) cells - with the goal of understanding the origins of the progenitor cells that produce adult-born neurons. Longstanding qualitative metrics for hemocyte classification were validated quantitatively. Then, in a longitudinal study, proliferation markers were used to label the hemocytes in vivo, followed by sampling the circulating hemocyte population over the course of two months. Hemolymph samples were taken at intervals to track the frequencies of the different hemocyte types. These data reveal sequential peaks in the relative frequencies of HCs, SGCs and GCs, which were identified using qualitative and quantitative measures. These findings suggest that the three hemocyte types comprise a single cellular lineage that occurs in the circulation, with each type as a sequential progressive stage in hemocyte maturation beginning with HCs and ending with GCs. When combined with previously published data, this timeline provides additional evidence that HCs serve as the primary neural progenitor during adult neurogenesis in P. clarkii.

Epigenetic variation in animal populations: Sources, extent, phenotypic implications, and ecological and evolutionary relevance.

Laboratory experiments and fieldwork with asexually reproducing invertebrates and vertebrates clearly revealed that animal populations can produce substantial phenotypic variation despite genetic identity. This epigenetically caused phenotypic variation comes from two different sources, namely directional environmental induction and bed-hedging developmental stochasticity. Both occur together and are mediated by molecular epigenetic mechanisms like DNA methylation, histone modifications and microRNAs. These epigenetic mechanisms are also involved in insect polyphenism, phenotypic changes in early domestication, and gene expression change and chromatin rearrangement during speciation. Epigenetic variation is particularly important for asexual populations helping them to stay in the game of life when the environmental conditions change. However, it is also relevant for sexually reproducing populations, as shown for genetically impoverished invasive groups, cave animals and sessile taxa that cannot evade unfavourable environmental conditions. Experiments revealed that epigenetic marks can be transgenerationally inherited and persist for several generations. First evidence suggests that inherited epimutations with phenotypic effects may end-up in phenotype-fixing genetic mutations by accelerated mutation of methylated nucleotides. Refined concepts, suitable animal models, fast and affordable new omics techniques that require only small tissue samples, and appropriate data interpretation tools are now available enabling future investigations in ecological and evolutionary epigenetics with high accuracy.

Sleep in the crustacean crayfish

Sleep is defined as a state of unconsciousness, reduced locomotive activity and rapid awakening, and is well established in mammals, birds, reptiles and teleosts. Commonly, it is also defined with electrical records (electroencephalogram), which are only well established in mammals and to some extent in birds. However, sleep states similar to those of mammals, except for electrical criteria, appear to occur in some invertebrates. Currently, the most compelling evidence of sleep in invertebrates has been obtained in the crayfish. In mammals, sleep is characterized by a brain state that is different from that of wakefulness, which includes a change to slow waves that has not been observed in insects. Herein, we show that the crayfish enters a brain state with a high threshold to vibratory stimuli, accompanied by a form of slow wave activity in the brain, quite different from that of wakefulness. Therefore, the crayfish can enter a state of sleep that is comparable to that of mammals.

Cytochrome P450 and Glutathione-S-Transferase Activity are Altered Following Environmentally Relevant Atrazine Exposures in Crayfish (Faxoniusvirilis).

The herbicide atrazine is heavily applied in the U.S. Midwest to control broadleaf weeds. It enters local streams and rivers through runoff and seepage, and exposure can affect non-target aquatic organisms, like crayfish. We examined sublethal effects of atrazine on the expression and activity of the detoxification enzymes cytochrome P450 (CYP450) and glutathione-S-transferase (GST) in crayfish. Crayfish were exposed to 0, 10, 40, 80, 100 and 300 ppb atrazine for 1, 2, 4, 7 and 10 days. Their hepatopancreas was collected and CYP450 expression and GST activity was analyzed. Atrazine exposure caused differential expression and activity of CYP450 and GST. CYP450 expression varied over exposure concentrations and time. Further, GST activity significantly increased following a 2 day, 10 ppb exposure to atrazine and a 300 ppb atrazine exposure for all days tested. We found that atrazine detoxification is a dynamic process that changes with the length and intensity of atrazine exposure.

Transglutaminase inhibition stimulates hematopoiesis and reduces aggressive behavior of crayfish, Pacifastacus leniusculus.

Transglutaminase (TGase) is a Ca2+-dependent cross-linking enzyme, which has both enzymatic and nonenzymatic properties. TGase is involved in several cellular activities, including adhesion, migration, survival, apoptosis, and extracellular matrix (ECM) organization. In this study, we focused on the role of the TGase enzyme in controlling hematopoiesis in the crayfish, Pacifastacus leniusculus We hypothesized that a high TGase activity could mediate an interaction of progenitor cells with the ECM to maintain cells in an undifferentiated stage in the hematopoietic tissue (HPT). We found here that the reversible inhibitor cystamine decreases the enzymatic activity of TGase from crayfish HPT, as well as from guinea pig, in a concentration-dependent manner. Cystamine injection decreased TGase activity in HPT without affecting production of reactive oxygen species. Moreover, the decrease in TGase activity in the HPT increased the number of circulating hemocytes. Interestingly the cystamine-mediated TGase inhibition reduced aggressive behavior and movement in crayfish. In conclusion, we show that cystamine-mediated TGase inhibition directly releases HPT progenitor cells from the HPT into the peripheral circulation in the hemolymph and strongly reduces aggressive behavior in crayfish.

The effect of temperature on white spot disease progression in a crustacean, Pacifastacus leniusculus.

The effects of temperature on the progression of White Spot Disease (WSD) have been studied in the freshwater crayfish Pacifastacus leniusculus. In this study, we aimed to understand the reason for previously observed low mortalities with white spot syndrome virus (WSSV) infected crayfish at low temperatures. The susceptibility of freshwater crayfish to WSSV was studied at different temperatures. The mortality rate at 6 °C was zero, meanwhile the animals kept at 22 °C developed WSD symptoms and died in a few days after WSSV injections, however upon transfer of animals from 6 °C to 22 °C the mortality reached 100% indicating that the virus is not cleared at 6 °C. Moreover, the VP28 expression at 6 °C was significantly lower compared to animals kept at 22 °C. We injected animals with demecolcine, an inhibitor that arrests the cell cycle in metaphase, and observed a delayed mortality. Furthermore, the VP28 expression was found to be lower in these animals receiving both injections with WSSV and demecolcine since cell proliferation was inhibited by demecolcine. We quantified WSSV copy numbers and found that virus entry was blocked at 6 °C, but not in demecolcine treatments. We supported this result by quantifying the expression of a clip domain serine protease (PlcSP) which plays an important role for WSSV binding, and we found that the PlcSP expression was inhibited at 6 °C. Therefore, our hypothesis is that the WSSV needs proliferating cells to replicate, and an optimum temperature to enter the host hematopoietic stem cells successfully.

Clonal genome evolution and rapid invasive spread of the marbled crayfish.

The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarus fallax, and also established a triploid AA'B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.

Characterization of a hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus.

Hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus has a distinct role from that of its homolog PmHHAP in the shrimp Penaeus monodon. Knockdown of PlHHAP in vitro using double-stranded RNA (dsRNA) had no effect on the cell morphology of hematopoietic tissue (HPT) cells. The total hemocyte number and caspase activity were unchanged after PlHHAP knockdown in vivo, in contrast to the results found in shrimp. Moreover, suppression of PlHHAP both in vitro and in vivo did not change the mRNA levels of some genes involved in hematopoiesis and hemocyte homeostasis. Interestingly, bacterial count and scanning electron microscope revealed that depletion of PlHHAP in intestine by RNAi resulted in higher number of bacteria in the crayfish intestine. Together, these results suggest that PlHHAP is not involved in hemocyte homeostasis in the crayfish P. leniusculus but appears to affect the bacterial number in the intestine through an unknown mechanism. Since PlHHAP has different functions from PmHHAP, we therefore named it HHAP-like protein.

The Behavior of Chromosomes During Parthenogenetic Oogenesis in Marmorkrebs Procambarus fallax f. virginalis.

Parthenogenetic oogenesis varies among and even within species. Based on cytological mechanisms, it can largely be divided into apomixis (ameiotic parthenogenesis) producing genetically identical progeny, and automixis (meiotic parthenogenesis) producing genetically non-identical progeny. Polyploidy is common in parthenogenetic species, although the association between parthenogenesis and polyploidy throughout evolution is poorly understood. Marmorkrebs, or the marbled crayfish, was first identified as a parthenogenetic decapod and was tentatively named as Procambarus fallax f. virginalis. Previous studies revealed that Marmorkrebs is triploid and produces genetically identical offspring, suggesting that apomixis occurs during parthenogenetic oogenesis. However, the behavior of chromosomes during the process of oogenesis is still not well characterized. In this study, we observed parthenogenetic oogenesis around the time of ovulation in P. fallax f. virginalis by histology and immunohistochemistry. During oogenesis, the chromosomes were separated into two groups and behaved independently from each other, and one complete division corresponding to mitosis (the second meiosis-like division) was observed. This suggests that parthenogenetic oogenesis in Marmorkrebs exhibits gonomery, a phenomenon commonly found in apomictic parthenogenesis in polyploid animals.

Cloning and molecular characterization of a putative voltage-gated sodium channel gene in the crayfish.

Voltage-gated sodium channel genes and associated proteins have been cloned and studied in many mammalian and invertebrate species. However, there is no data available about the sodium channel gene(s) in the crayfish, although the animal has frequently been used as a model to investigate various aspects of neural cellular and circuit function. In the present work, by using RNA extracts from crayfish abdominal ganglia samples, the complete open reading frame of a putative sodium channel gene has firstly been cloned and molecular properties of the associated peptide have been analyzed. The open reading frame of the gene has a length of 5793 bp that encodes for the synthesis of a peptide, with 1930 amino acids, that is 82% similar to the α-peptide of a sodium channel in a neighboring species, Cancer borealis. The transmembrane topology analysis of the crayfish peptide indicated a pattern of four folding domains with several transmembrane segments, as observed in other known voltage-gated sodium channels. Upon analysis of the obtained sequence, functional regions of the putative sodium channel responsible for the selectivity filter, inactivation gate, voltage sensor, and phosphorylation have been predicted. The expression level of the putative sodium channel gene, as defined by a qPCR method, was measured and found to be the highest in nervous tissue.

Long-Term Starvation and Posterior Feeding Effects on Biochemical and Physiological Responses of Midgut Gland of Cherax quadricarinatus Juveniles (Parastacidae).

We investigated the effect of long-term starvation and posterior feeding on energetic reserves, oxidative stress, digestive enzymes, and histology of C. quadricarinatus midgut gland. The crayfish (6.27 g) were randomly assigned to one of three feeding protocols: continuous feeding throughout 80 day, continuous starvation until 80 day, and continuous starvation throughout 50 day and then feeding for the following 30 days. Juveniles from each protocol were weighed, and sacrificed at day 15, 30, 50 or 80. The lipids, glycogen, reduced glutathione (GSH), soluble protein, lipid peroxidation (TBARS), protein oxidation (PO), catalase (CAT), lipase and proteinase activities, and histology were measured on midgut gland. Starved crayfish had a lower hepatosomatic index, number of molts, specific growth rate, lipids, glycogen, and GSH levels than fed animals at all assay times. The starvation did not affect the soluble protein, TBARS, PO levels and CAT. In starved juveniles the lipase activity decreased as starvation time increased, whereas proteinase activity decreased only at day 80. The histological analysis of the starved animals showed several signs of structural alterations. After 30 days of feeding, the starved-feeding animals exhibited a striking recovery of hepatosomatic index, number of molts, lipids and glycogen, GSH, lipase activity and midgut gland structure.

White spot syndrome virus enters crayfish hematopoietic tissue cells via clathrin-mediated endocytosis.

White spot syndrome virus (WSSV) is a major pathogen of aquacultured shrimp. However, the mechanism of its entry remains poorly understood. In this study, by analyzing the internalization of WSSV using crayfish hematopoietic tissue (HPT) cells, we showed that WSSV virions were engulfed by cell membrane invaginations sharing the features of clathrin-coated pits and then internalized into coated cytoplasmic vesicles. Further investigation indicated that WSSV internalization was significantly inhibited by chlorpromazine (CPZ) but not genistein. The internalized virions were colocalized with endogenous clathrin as well as transferrin which undergoes clathrin-dependent uptake. Preventing endosome acidification by ammonium chloride (NH4Cl) or chloroquine (CQ) dramatically reduced WSSV entry as well. Moreover, disturbance of dynamin activity or depletion of membrane cholesterol also blocked WSSV uptake. These data indicate that WSSV enters crayfish HPT cells via clathrin-mediated endocytosis in a pH-dependent manner, and membrane cholesterol as well as dynamin is critical for efficient viral entry.

The Effects of Biodiesel and Crude Oil on the Foraging Behavior of Rusty Crayfish, Orconectes rusticus.

Environmental pollutants, such as crude oil and other petroleum-based fuels, inhibit and limit an organism's ability to perceive a chemical stimulus. Despite the increased use of alternative fuels, such as biodiesel, there have been few studies investigating the impact of these chemicals on the behavior of aquatic organisms. The purpose of this study was to compare the sublethal effects of biodiesel and crude oil exposure on chemically mediated behaviors in a freshwater keystone species. Crayfish (Orconectes rusticus) were tested on their ability to respond appropriately to a positive chemical stimulus within a Y-maze choice paradigm. Behavior was quantified by measuring time spent finding an odor source, duration of time spent at the odor source, percentage of crayfish that found the odor source, and percentage of crayfish that chose the correct arm of the arena. Results indicated negative impacts of both biodiesel and crude oil on the ability of crayfish to locate the food source. However, there were no significant differences between behavioral performances when crayfish were exposed to crude oil compared with biodiesel. Thus, biodiesel and crude oil have equally negative effects on the chemosensory behavior of crayfish. These findings indicate that biodiesel has the potential to have similar negative ecological impacts as other fuel source toxins.

Protection of the Crayfish Mechanoreceptor Neuron and Glial Cells from Photooxidative Injury by Modulators of Diverse Signal Transduction Pathways.

Oxidative stress is the reason of diverse neuropathological processes. Photodynamic therapy (PDT), an effective inducer of oxidative stress, is used for cancer treatment, including brain tumors. We studied the role of various signaling pathways in photodynamic injury and protection of single neurons and satellite glial cells in the isolated crayfish mechanoreceptor. It was photosensitized with alumophthalocyanine Photosens in the presence of inhibitors or activators of various signaling proteins. PDT eliminated neuronal activity and killed neurons and glial cells. Inhibitory analysis showed the involvement of protein kinases Akt, glycogen synthase kinase-3ß (GSK-3ß), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinases 1 and 2 (MEK1/2), calmodulin, calmodulin-dependent kinase II (CaMKII), adenylate cyclase, and nuclear factor NF-κB in PDT-induced necrosis of neurons. Nitric oxide (NO) and glial cell-derived neurotrophic factor (GDNF) reduced neuronal necrosis. In glial cells, protein kinases Akt, calmodulin, and CaMKII; protein kinases C and G, adenylate cyclase, and p38; and nuclear transcription factor NF-κB also mediated PDT-induced necrosis. In contrast, NO and neurotrophic factors nerve growth factor (NGF) and GDNF demonstrated anti-necrotic activity. Phospholipase Cγ, protein kinase C, GSK-3ß, mTOR, NF-κB, mitochondrial permeability transition pores, and NO synthase mediated PDT-induced apoptosis of glial cells, whereas protein kinase A, tyrosine phosphatases, and neurotrophic factors NGF, GDNF, and neurturin were involved in protecting glial cells from photoinduced apoptosis. Signaling pathways that control cell survival and death differed in neurons and glia. Inhibitors or activators of some signaling pathways may be used as potential protectors of neurons and glia from photooxidative stress and following death.

Using biochemical markers to assess the effects of imposed temperature stress on freshwater decapod crustaceans: Cherax quadricarinatus as a test case.

The effects of thermal stress can impact negatively on the abundance and distribution of temperature-sensitive species, particularly freshwater crustaceans. This study investigated the effects of thermal stress on physiological and biochemical parameters at five treatment temperatures resulting in minimal (25 °C), moderate (27, 29 °C) or severe (31, 33 °C) thermal stress in the common tropical freshwater crayfish Cherax quadricarinatus. The aim was to develop a suite of stress-sensitive assays to use on threatened populations of freshwater crustaceans, particularly those restricted to cooler temperatures and only found in high altitude refugia. Significant increases in indicators of oxidative and metabolic stress were observed at 29 °C and were elevated further at 33 °C. After a 50-day acclimation to an imposed temperature stress, significant changes in the level of total glutathione, total lipids, muscular protein, total haemocyte count, lipid peroxidation and protein carbonyls were observed between treatments while superoxide dismutase activity and haemolymph protein concentrations did not change. The data provided proof of concept that measuring key biochemical responses to high temperature can provide a means of contrasting the level of thermal stress experienced between individuals of the same species adapted to different temperatures. The methods developed are expected to be of use in research on wild populations of other freshwater poikilothermic organisms, particularly those susceptible to increased environmental temperatures associated with climate change.

Different transcription regulation routes are exerted by L- and D-amino acid enantiomers of peptide hormones.

Conversion of one or more amino acids in eukaryotic peptides to the D-enantiomer configuration is catalyzed by specific L/D-peptide isomerases and it is a poorly investigated post-translational modification. No common modified amino acid or specific modified position has been recognized, and mechanisms underlying changes in the peptide function provided by this conversion are not widely studied. The 72 amino acid crustacean hyperglycemic hormone (CHH) in Astacidea crustaceans exhibits a co-existence of two peptide enantiomers with either D- or L-phenylalanine as their third residue. It is a pleiotropic hormone regulating several physiological processes in different target tissues and along different time scales. CHH enantiomers differently affect time courses and intensities of examined processes. The short-term effects of the two isomers on gene expression were examined in the hepatopancreas, gills, hemocytes and muscles of the astacid Pontastacus leptodactylus. Gene expression in muscles and hemocytes was not affected by either of the isomers. Two modes of action for CHH were elucidated in the hepatopancreas and the gills: specific gene induction in both organs by D-CHH, and targeted attenuation caused by both enantiomers in the gills. Consequently, a two-receptor system is proposed for conveying the effect of the two CHH isomers.

Acute toxicity of triazine pesticides to juvenile signal crayfish (Pacifastacus leniusculus).

OBJECTIVES: Crustacea are at high risk of toxic effects of agricultural pesticides. Currently, many questions regarding the toxicity of triazine herbicides to crayfish remain unresolved. The aim of this research was to evaluate the acute toxicity of atrazine, hexazinone, metribuzine, prometryne, simazine, and terbutryne to juvenile signal crayfish (Pacifastacus leniusculus). DESIGN: Acute toxicity tests were performed in accordance with standardized guidelines for testing of chemicals, OECD no. 203, using a semistatic test system. Signal crayfish juveniles (n=672) of 49.0-81.5 mg weight and 12.8-16.0 mm total length were used for the bioassay. Mortalities were recorded daily to 96 h. Each pesticide was tested at concentrations of 1, 10, 40, 70, and 100 mg.l-1. Percent mortalities were analyzed by linear regression, and median lethal concentration (LC50) values were computed using probit analysis EKO-TOX 5.2 software. RESULTS: 96hLC50 values for juvenile signal crayfish were 12.1 mg.l-1 for atrazine, 13.9 mg.l-1 for terbutryne, 14.4 mg.l-1 for prometryne, 19.5 mg.l-1 for hexazinone, 30.6 mg.l-1 for metribuzine, and 77.9 mg.l-1 for simazine. Atrazine showed the greatest toxicity to signal crayfish. CONCLUSIONS: The present study demonstrated that triazines are toxic to signal crayfish. Signal crayfish is more sensitive than the fish for atrazine, hexazine, metribuzine, and for these triazines signal crayfish can be used as a bio-indicator of environmental contamination.

Adult neurogenesis in the crayfish brain: the hematopoietic anterior proliferation center has direct access to the brain and stem cell niche.

Neuronal stem cells residing in a niche on the surface of the adult crayfish (Procambarus clarkii) brain are not self-renewing. However, the neuronal precursors in the niche are not depleted despite continued neurogenesis and the exit of precursor cells from the niche throughout the organism's life. The neurogenic niche is therefore not a closed system, and we have previously proposed that the stem cell pool is replenished from the hematopoietic system. Noonin et al. (2012) demonstrated that the hematopoietic system in the crayfish Pacifastacus leniusculus includes an anterior proliferation center (APC) lying near the brain; they suggest that multipotent stem cells are concentrated in this region, and that the APC may provide neuronal stem cells for adult neurogenesis. The present study extends this work by describing the location and cellular organization of hematopoietic tissues in P. clarkii. We find that the APC lies within the cor frontale, or auxiliary heart, which pumps hemolymph to the brain and eyes through the cerebral and ophthalmic arteries, respectively. Vascular extensions of the cerebral artery converge on the neurogenic niche. APC cells lie in layered sheets within the cor frontale and form rosette-like structures reminiscent of stem cells in other developing tissues. We confirm here that APC cells in P. clarkii have characteristics of multipotent stem cells, and that their location within the cor frontale allows direct access to regions in the central nervous system in which adult neurogenesis occurs.

Invertebrate hematopoiesis: an anterior proliferation center as a link between the hematopoietic tissue and the brain.

During evolution, the innate and adaptive immune systems were developed to protect organisms from non-self substances. The innate immune system is phylogenetically more ancient and is present in most multicellular organisms, whereas adaptive responses are restricted to vertebrates. Arthropods lack the blood cells of the lymphoid lineage and oxygen-carrying erythrocytes, making them suitable model animals for studying the regulation of the blood cells of the innate immune system. Many crustaceans have a long life span and need to continuously synthesize blood cells, in contrast to many insects. The hematopoietic tissue (HPT) of Pacifastacus leniusculus provides a simple model for studying hematopoiesis, because the tissue can be isolated, and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. Here, we demonstrate new findings of a physical link between the HPT and the brain. Actively proliferating cells were localized to an anterior proliferation center (APC) in the anterior part of the tissue near the area linking the HPT to the brain, whereas more differentiated cells were detected in the posterior part. The central areas of HPT expand in response to lipopolysaccharide-induced blood loss. Cells isolated from the APC divide rapidly and form cell clusters in vitro; conversely, the cells from the remaining HPT form monolayers, and they can be induced to differentiate in vitro. Our findings offer an opportunity to learn more about invertebrate hematopoiesis and its connection to the central nervous system, thereby obtaining new information about the evolution of different blood and nerve cell lineages.

Morphine-conditioned cue alters c-Fos protein expression in the brain of crayfish.

With a highly organized stereotypic behavior and a simplified neuronal system that is characterized by cellular modularity, crayfish (Orconectes rusticus) represents an excellent model that we used in this study to explore how a drug-conditioned-cue alters c-Fos protein expression in the brain of an invertebrate species. The first set of experiments revealed that a single injection of different doses of morphine (3.0 µg/g, 6.0 µg/g and 12.0 µg/g) into the circulatory system of crayfish significantly increased locomotor activity. Repeated injections of morphine increased locomotion at lower doses (3.0 µg/g and 6.0 µg/g), and decreased locomotion at a higher dose of 12.0 µg/g. The second experiment revealed that a repeated or single injection of morphine serves as reward when paired with a distinct visual environment. In the third experiment, we found that the c-Fos profile of morphine treated crayfish in an unconditioned environment did not show a significant increase from the basal level comparable to saline treated crayfish. The brains of crayfish were more active during exposure to the cue-elicited drug conditioned environment than the unconditioned environment. These results indicate that chronic morphine treatment alone is not sufficient to induce changes in the expression of c-Fos; instead, morphine-environment pairing in a specific context contributes to the expression of alterations in c-Fos regulation. The enhancement of c-Fos expression in the brain of crayfish seems to reflect the sensory or anticipatory facets of conditioning that suggests that potential and even unanticipated hypotheses in drug addiction can emerge from studies of addiction in crayfish.