System Of Crayfish Research Articles

Dopamine receptor (DAR) and dopa decarboxylase (DDC) mediate hepatopancreas antibacterial innate immune reactions in Procambarus clarkii

Dopa is decarboxylated by dopa decarboxylase (DDC) to form dopamine, which is a significant signaling molecule in the neuroendocrine system. The dopamine receptor (DAR) is an important transmembrane receptor responsible for receiving extracellular signals in the DAR-mediated signaling pathway. In the present study, the expression patterns of Pc-dar were investigated after bacterial challenge. The obviously changed expression patterns showed Pc-dar was related to the antibacterial innate immunity. Endogenous Pc-DDC enzymatic activities were obviously downregulated after Pc-ddc dsRNA injection. The expression level of Pc-dar mRNA was obviously upregulated after bacterial injection when the expression level of Pc-ddc was knocked down. In addition, the upregulation trend of endogenous Pc-DDC enzymatic activities was obviously restrained after bacterial stimulation when Pc-ddc was knocked down. Finally, melanization was downregulated in crayfish hemolymph compared with the dsGFP injection group. In the RNAi assay, the results of qRT-PCR showed that Toll (TLRs) signaling pathway-related genes were activated in the early stages of bacterial stimulation when Pc-ddc was knocked down. Four tested ROS-related antioxidant enzyme genes were significantly upregulated after bacterial challenge compared with the dsGFP injection group. The above results indicated that Pc-DDC and Pc-DAR play important mediating roles in the neuroendocrine immune (NEI) system of crayfish.

High degree of non-genetic phenotypic variation in the vascular system of crayfish: a discussion of possible causes and implications

In this study, the hemolymph vascular system (HVS) in two cambarid crayfishes, i.e. the Marbled Crayfish, Procambarus virginalis Lyko, 2017 and the Spiny Cheek Crayfish, Faxonius limosus (Rafinesque, 1817), is investigated in regard of areas of non-genetic phenotypic variation. Despite their genetic identity, specimens of P. virginalis show variability in certain features of the HVS. Thus, we describe varying branching patterns, sporadic anastomoses, and different symmetry states in the vascular system of the marbled crayfish. We visualize our findings by application of classical and modern morphological methods, e.g. injection of casting resin, micro-computed tomography and scanning electron microscopy. By comparing our findings for P. virginalis to the vasculature in sexually reproducing crayfishes, i.e. F. limosus and Astacus astacus, we discuss phenotypic variation of the HVS in arthropods in general. We conclude that constant features of the HVS are hereditary, whereas varying states identified by study of the clonal P. virginalis must be caused by non-genetic factors and, that congruent variations in sexually reproducing F. limosus and A. astacus are likely also non-genetic phenotypic variations. Both common causal factors for non-genetic phenotypic variation, i.e., phenotypic plasticity and stochastic developmental variation are discussed along our findings regarding the vascular systems. Further aspects, such as the significance of non-genetic phenotypic variation for phylogenetic interpretations are discussed.

Lactobacillus acidophilus and L. plantarum improve health status, modulate gut microbiota and innate immune response of marron (Cherax cainii)

This study aimed to investigate the combined effects of two most potent probiotic bacteria Lactobacillus acidophilus and Lactobacillus plantarum on overall health and immune status of freshwater crayfish, marron under laboratory conditions. A total of 36 marron were distributed into six different tanks and two different feeding groups, control and probiotic-fed group. After acclimation, control group was fed with basal diet while probiotic group was fed 109 CFU/mL per kg of bacterial supplemented feed for 60 days. The results showed no significant differences in weight gain, however, probiotic feed significantly enhanced some hemolymph parameters and biochemical composition of tail muscle. Histology data revealed better hepatopancreas health and higher microvilli counts in the marron gut fed probiotic diet. The probiotic bacteria triggered significant shift of microbial communities at different taxa level, mostly those reported as beneficial for crayfish. The probiotic diet also enriched the metabolic functions and genes associated with innate immune response of crayfish. Further correlation analysis revealed significant association of some taxa with increased activity for hemolymph and immune genes. Therefore, dietary Lactobacillus supplementation can modulate the overall health and immunity as well as gut microbial composition and interaction network between gut microbiota and immune system in crayfish.

Molecular characterization and expression analysis of dopa decarboxylase involved in the antibacterial innate immunity of the freshwater crayfish, Procambarus clarkii

Dopa decarboxylase (DDC) is responsible for the synthesis of dopamine, which acts as an important modulator in the nervous systems of vertebrates and invertebrates. Recent studies have indicated that DDC also plays crucial roles in the insect innate immune system. However, the functions of DDC in immunomodulation in crustaceans have not been thoroughly elucidated to date. In this study, a new full-length cDNA of the DDC protein was identified from red swamp crayfish, Procambarus clarkii (named Pc-ddc). The ORF of Pc-ddc encoded 474 amino acids, which possessed a 377-amino-acid domain. Pc-ddc was expressed at a relatively high level in the hemocytes and gills of crayfish. This protein was expressed at a relatively low level in the hepatopancreas and intestine. The expression level of Pc-ddc was clearly upregulated in hemocytes, hepatopancreas, gills, and intestine tissues after challenge with S. aureus or E. ictaluri. The results of the enzyme catalysis assay showed that the enzyme catalysis activity of rPc-DDC was 35 ± 2.8 ng h−1 mg−1 (n = 3). In addition, the results of the mimetic crayfish hemocytes encapsulation assay showed that the encapsulation rate of beads coated with rPc-DDC was clearly increased. The results of the bacterial binding assay showed that rPc-DDC strongly binds to S. aureus and E. ictaluri. Finally, when Pc-ddc was knocked down, the number of surviving crayfish clearly decreased after S. aureus or E. ictaluri was injected. All of these results indicate that Pc-DDC is an important immunomodulating enzyme in the neuroendocrine-immune (NEI) system of crayfish.

Involvement of Melatonin in the Regulation of the Circadian System in Crayfish.

Melatonin (MEL) is an ancient molecule, broadly distributed in nature from unicellular to multicellular species. MEL is an indoleamine that acts on a wide variety of cellular targets regulating different physiological functions. This review is focused on the role played by this molecule in the regulation of the circadian rhythms in crayfish. In these species, information about internal and external time progression might be transmitted by the periodical release of MEL and other endocrine signals acting through the pacemaker. We describe documented and original evidence in support of this hypothesis that also suggests that the rhythmic release of MEL contributes to the reinforcement of the temporal organization of nocturnal or diurnal circadian oscillators. Finally, we discuss how MEL might coordinate functions that converge in the performance of complex behaviors, such as the agonistic responses to establish social dominance status in Procambarus clarkii and the burrowing behavior in the secondary digging crayfish P. acanthophorus.

Circadian rhythm in melatonin release as a mechanism to reinforce the temporal organization of the circadian system in crayfish.

Melatonin (MEL) is a conserved molecule with respect to its synthesis pathway and functions. In crayfish, MEL content in eyestalks (Ey) increases at night under the photoperiod, and this indoleamine synchronizes the circadian rhythm of electroretinogram amplitude, which is expressed by retinas and controlled by the cerebroid ganglion (CG). The aim of this study was to determine whether MEL content in eyestalks and CG or circulating MEL in hemolymph (He) follows a circadian rhythm under a free-running condition; in addition, it was tested whether MEL might directly influence the spontaneous electrical activity of the CG. Crayfish were maintained under constant darkness and temperature, a condition suitable for studying the intrinsic properties of circadian systems. MEL was quantified in samples obtained from He, Ey, and CG by means of an enzyme-linked immunosorbent assay, and the effect of exogenous MEL on CG spontaneous activity was evaluated by electrophysiological recording. Variation of MEL content in He, Ey, and CG followed a circadian rhythm that peaked at the same circadian time (CT). In addition, a single dose of MEL injected into the crayfish at different CTs reduced the level of spontaneous electrical activity in the CG. Results suggest that the circadian increase in MEL content directly affects the CG, reducing its spontaneous electrical activity, and that MEL might act as a periodical signal to reinforce the organization of the circadian system in crayfish.

English

Abstract Recent studies have revealed integral and complex relationships between hosts and their symbionts. Some of these findings demonstrate that symbionts can affect the gene expression of their hosts. We used a model cleaning symbiosis system of crayfish and their branchiobdellidan symbionts to examine whether symbionts could affect host gene expression through indirect means, by changing the interaction of the host crayfish with the environment. Previous research has shown this symbiosis to be a complex, context dependent relationship in which outcomes can shift between mutualism, when branchiobdellidans act as cleaners of their hosts, and parasitism, when damage to hosts’ gills through branchiobdellidan feeding produces negative effects. These shifts are known to occur with changes in symbiont densities and environmental conditions. We manipulated densities of branchiobdellidans on host crayfish and examined the effect on expression of 3 candidate immune genes. We had 2 competing working hypotheses: 1) That branchiobdellidans would cause an increase expression in immune genes because of damage to the hosts’ gills; 2) That branchiobdellidans would decrease expression of immune genes by acting as cleaners and thus alleviating environmental stress. We found that the second hypothesis was strongly supported when branchiobdellidan activity decreased expression in 2 of the 3 candidate genes. One of these genes (astacidin) was related to generalized immune defense, while the other (prophenoloxidase) is a key part of the melanization cascade that provides defense in the case of physical damage and intrusion of microbes or foreign bodies. Given that there is no known mechanism through which branchiobdellidans can directly affect host physiology through chemical or genetic interactions, we concluded that this alteration in gene expression occurred through alleviation of environmental stress by the symbionts. Such indirect effects may be common in nature and continued study using easily manipulated systems like the crayfish-branchiobdellidan symbiosis may continue to produce insights regarding the importance of symbioses in ecological systems.

The rewarding properties of methamphetamine in an invertebrate model of drug addiction

The rewarding properties of drugs in the mammalian system depend on their ability to activate appetitive motivational states. The associated underlying mechanism is strongly conserved in evolution and invertebrates have recently emerged as a powerful new model in addiction research. The natural reward system in crayfish has surprisingly proven sensitive to human drugs of abuse, providing a new model for research into the basic biological mechanisms of drug addiction. In this study, we examined the presence of natural reward systems in crayfish, and then characterized its sensitivity to 2.5μg/g, 5.0μg/g and 10.0μg/g doses of methamphetamine (METH). Using the conditioned place preference (CPP) paradigm, we demonstrated that irrespective of the number of doses of METH injected into the pericardial system, crayfish seek out a particular tactile environment that had previously been paired with the METH. This study demonstrates that crayfish offer a comparative and complementary approach in addiction research. It contributes an evolutionary context to our understanding of a key component in learning and of natural reward as an important life-sustaining process.

Characterization of a C-type lectin (PcLec2) as an upstream detector in the prophenoloxidase activating system of red swamp crayfish

C-type lectins are abundantly found in crustaceans. They function in the immune system by recognizing, opsonizing, or agglutinating. Some of them also feature anti-microbial activity. In this study, we identify a hepatopancreas-specific C-type lectin (PcLec2) that responds significantly to immune challenges in red swamp crayfish ( Procambarus clarkii). Recombinant PcLec2 shows no agglutinating or anti-microbial activity. It can bind to lipopolysaccharides and bacterial cells in the absence of calcium, and its binding to Gram-negative bacteria is stronger than that to Gram-positive ones. Moreover, the protein can stimulate the activation of prophenoloxidase both in vitro and in vivo. We conclude that this C-type lectin may be an upstream detector of the prophenoloxidase activating system in crayfish.

Development, growth, and plasticity in the crayfish olfactory system.

Decapod crustaceans have a well-defined olfactory system characterised by a set of chemosensitive sensilla grouped together in an array (the olfactory organ) on their antennules. Olfactory receptor neurons in the olfactory organ project exclusively to, and terminate in, cone-shaped olfactory glomeruli in a discrete neuropil in the brain, the olfactory lobe. The olfactory organ appears to be the only afferent input to the olfactory lobe, making the system convenient for the study of its development and growth. The progression of development of the olfactory system is a continuum and can be traced from the first appearance of peripheral receptor cells and central stem cells through to the construction of the tracts and neuropils that constitute the adult system. Cell proliferation leading to the production of peripheral and central olfactory neurons can be observed with mitotic markers in both embryonic stages and in postembryonic growth. Cell proliferation in the olfactory system in crayfish persists throughout the lives of the animals and can be modulated by manipulating the living conditions imposed on growing animals. Large serotonergic neurons that are associated with the olfactory system may play a role in the regulation of cell proliferation.

A Lipopolysaccharide- and β-1,3-Glucan-binding Protein from Hemocytes of the Freshwater Crayfish Pacifastacus leniusculus: PURIFICATION, CHARACTERIZATION, AND cDNA CLONING

A lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to beta-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and beta-1, 3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the beta-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.

Regulation of the Cardiovascular System in Crayfish

Regulation of the Cardiovascular System in Crayfish

Cholinergic modulation of the swimmeret motor system in crayfish.

1. The muscarinic agonist pilocarpine induced the swimmeret motor pattern in resting isolated preparations of the crayfish abdominal nerve cord and modulated the burst frequency in a dose-dependent manner. 2. Nicotine did not elicit rhythmic activity in resting isolated preparations but increased the burst frequency in active preparations. Nicotine produced higher burst frequencies than pilocarpine. 3. The acetylcholine (ACh) analogue carbachol combined the effects of pilocarpine and nicotine. It activated isolated resting preparations and increased the burst frequency as effectively as nicotine. The ACh-esterase inhibitor eserine also increased the burst frequency in active preparations. 4. Neither muscarinic nor nicotinic antagonists disrupted the proctolin-induced motor pattern, suggesting that proctolin and cholinergic agonists affect two different pathways for the activation of the swimmeret system. 5. We conclude that cholinergic interneurons participate in initiation of the swimmeret motor pattern and can modulate its burst frequency.

The crayfish pathogen Psorospermium haeckeli activates the prophenoloxidase activating system of freshwater crayfish in vitro

Preparations of the crayfish pathogen Psorospermium haeckeli were found to activate the prophenoloxidase activating (proPO) system in haemocyte lysates of two crayfish species Pacifastacus leniusculus and Astacus astacus. This is the first report showing that this parasite can elicit the activation of a defence system in crayfish and that the activation appears to be similar to the activation of the proPO system triggered by different microbial polysaccharides.

Source and nature of disturbance-chemical system in crayfish

The responses of crayfish to water from aquaria containing either undisturbed or disturbed animals were observed. The crayfishOrconectes propinquus andO. rusticus showed no response to disturbed-conspecific water. Individuals ofO. virilis respond not only to disturbed crayfish but to other taxa (the leechMacrobdella decora, the darterEtheostoma exile, and rock bassAmbloplites rupestris), but not to the painted turtleChrsymes picta. Additional tests indicated partial responses byO. virilis to ammonium and to a chemical or chemicals released from the green gland of crayfish. Ablation experiments indicated the antennules as the site of reception of the chemicals. Additional behavioral tests indicated that detection of the disturbance chemical(s) results in the crayfish showing low-level alert for more than 15 min, once an initial priming period has passed.

Unexpected divergence among identified interneurons in different abdominal segments of the crayfish Procambarus clarkii

The command elements that initiate and coordinate the abdominal movements in crayfish show little similarity between the various abdominal segments. Our criteria for similarity among interneurons were based on both cell morphology and electrophysiology. By contrast, previously published evidence shows much greater intersegmental similarity in the skeletal, muscular, motoneuronal, and sensory components of the abdominal system in crayfish, structures that are controlled by or send information to the command elements. Therefore, unlike the command elements, these structures have retained nearly identical form and function in the various segments. We also found in different ganglia examples of interneurons involved with abdominal positioning behavior that have similar morphology but different function and vice versa. Such interneurons could represent divergent pairs of serial homologues. It is unknown why so many of the abdominal positioning interneurons have become different. The various ganglia may perform subtly different functions, requiring differences in the positioning interneurons but not in the motor neurons or muscles. Alternatively, some of the abdominal positioning interneurons underlie more than one behavior; consequently, selection acting on these multiple functions may have changed these interneurons through evolution.

Rhodopsin-porphoropsin system in crayfish

s 1699 RHODOPSIN-PORPHYROPSIN SYSTEM IN CRAYFISH TATSUO SUZUKI and MOMOYO MAKINO-TASAKA Department of Pharmacology, Hyogo College of Medicine, Nishinomiya, Hyogo 663, Japan A sensitive analytical method was developed to study the rhodopsin-porphysopsin system in the eye. Oximes of 11-cis-retinal, 11-cis-3-dehydroretinal, all-trans-retinal, and all-trans-3-dehydroretinal were quantitatively determined by high-pressure liquid chromatography. The rhodopsin and porphyropsin of bullfrog retina were analyzed with this method. The results agreed with those obtained from the bleaching kinetics of visual pigments extracted with detergent. The analytical method was so sensitive that a 1/500 part of a bullfrog retina was sufficient for analysis. We studied the chromophore of the visual pigment of freshwater crustacean with this analytical method. The dark-adapted eye of crayfish (Procambarus clarkii) contained 11-cis-3-dehydroretinal which was isomerized to all-trans form by light-adaptation. All-trans-isomers produced by light were slowly reconverted to 11-cis-isomers in the dark with a half-time of 20 h (20°C). Seasonal variation was observed in the 3-dehydroretinal content. The value was below 5~ in summer and about 40~ in winter. The results suggest that the crayfish has a rhodopsin-porphyropsin system similar to that in freshwater fish. LOCALIZATION OF DIFFERENT VISUAL PIGMENTS WITHIN THE RHABDOMS OF INSECT COMPOUND EYES HELMUT LANGER Institute for Animal Physiology, Ruhr-University, P.O. Box 10 21 48, D-4630 Boehum 1, F.R.G. After identification of visual pigments in extracts from insect eyes, there remains the problem of their distribution between the different types of visual cells within the ommatidium. Compound eyes after illumination with monochromatic light were investigated by microspectrophotometry and electron microscopy. Photometrically, difference spectra could be obtained from single rhabdomeres of open rhabdoms, indicating only one receptor pigment within a rhabdomere. In fused rhabdoms, distinct changes of the microvillar structure were found in the rhabdomeres of different receptor cells after different monochromatic illuminations. The changes were proved to occur in vivo only temporarily. Examples presented are the eyes of hemiptera, especially of the pond skater, Gerris lacustris, with three pigments, and of nocturnal lepidoptera, especially of the armyworm moth, Spodoptera exempta, with four pigments. This work was done in collaboration with Drs B. Hamann, C. C. Meinecke, P. Schlecht, B. Welsch (Bochum), and Dr R. Schwind (University of Regensburg). It was financially supported by Deutsche Forschungsgemeinschaft. MICROSPECTROFLUOROMETRY ON FLY PHOTORECEPTORS IN VIVO. SPECTRAL PROPERTIES OF VISUAL PIGMENT FLUORESCENCE B. KRUIZINGA and D. G. STAVENGA Biophysics Department, Laboratorium voor Algemene Natuurkunde, Rijksuniversiteit Groningen, The Netherlands Visual pigment fluorescence has been studied in the compound eyes of houseflies Musca and blowflies Calliphora, white eyed mutants, in vivo. Rhodopsin R fluorescence is negligible, but the metarhodopsin state M fluoresces distinctly in the red. At extreme illumination intensities a 3-5 times stronger fluorescing M' state is populated. Both M and M' have emission spectra, peaking at about 660 nm, which do not noticeably change in the physiological temperature range, so, indicating the rigidity of the visual molecule in the membrane. The fluorescence life times of M and M', measured by applying a nitrogen pulse laser, are shorter than 40 nsec. Metarhodopsin M fluorescence has been used in a study of the photochemical cycle. The results were in good agreement with those obtained by transmission microspectrophotometry [Kruizinga et al. (1983), Biophys. Struct. Mech. 9, 299]. Microspectrofluorometry hence can be utilised for analysing photopigment processes in completely intact, living animals.

The prophenoloxidase activating system in crayfish

1. 1. A preparation (designated 0–40 fraction) containing stable prophenoloxidase (proPO) and other dormant components of the prophenoloxidase activating system was obtained from crayfish hemocytes. 2. 2. Activation of proPO in the 0–40 fraction was elicited by β1,3-glucans, SDS, trypsin or heat, while a protease inhibitor, p-NPGB, inhibited activation of proPO by β1,3-glucans but, not activation by SDS or heat. 3. 3. Ca 2+ was always necessary for the activation of proPO and treatment of the 0–40 fraction with EDTA caused irreversible inactivation of proPO activating system, seemingly leaving proPO intact. 4. 4. The enzyme responsible for activating proPO could be separated from proPO and this enzyme was found to be inhibited by p-NPGB. This enzyme could activate proPO in the 0–40 fraction treated with EDTA. 5. 5. Protease activity increased more than 10-fold in the 0–40 fraction after the incubation with β1,3-glucans and Ca 2+. 6. 6. The proPO activating system may operate as a recognition system in crayfish, and it is suggested that this system functions as a complement-like system in arthropods.

A quantitative approach to habituation and to evaluation of the effects of noisy neural pathways in a crayfish system.

The walk response to photostimulation of the crayfish caudal photoreceptor system is analyzed in terms of statistical dependence between input and output utilizing information theory. The analysis provides an identification of habituation and dishabituation in this system. Further, one of the dishabituatory responses can itself be seen to habituate and dishabituate. A quantitative measure of the statistical dependence of walk response on light stimulus intensity is found and compared with similar calculations presented previously for the ability of the neural pathways involved in this system to transmit information. It is shown that dispersion of interpulse intervals during axonal and synaptic transmission does not significantly limit the dependence of the behavioral walk response on the intensity of light stimulation.

STABILITY OF THE CHROMATOPHOROTROPINS OF THE DWARF CRAYFISH, CAMBARELLUS SHUFELDTI, AND THEIR EFFECTS ON ANOTHER CRAYFISH

The chromatophore systems of three crayfishes have been investigated to a considerable degree. Of these most is known about color changes in the dwarf crayfish, Cambarellus shufeldti. Brown and Meglitsch (1940) found that the sinus gland in the eyestalk of the crayfish Orconectes immunis contained a chromatophorotropin that concentrated red pigment. McVay (1942) demonstrated that the central nervous system of this crayfish produced a material that concentrated red pigment. The chromatophore system of the dwarf crayfish has been the subject of previous investigations by Fingerman (1957a, 1957b) and Fingerman and Lowe (1957a, 1957b). The red pigment dispersed maximally when dwarf crayfish were put on a black background and concentrated maximally when the crayfish were put on a white background. In the supraesophageal ganglia, circumesophageal connectives, and tissues of the eyestalks were chromatophorotropins that dispersed as well as concentrated red pigment (Fingerman, 1957a). Behavior of red pigment on isolated portions of the carapace of Cambarellus that had been on black and on white backgrounds was also investigated (Fingerman, 1957b). Red pigment had an inherent tendency to concentrate nearly maximally when the chromatophores were no longer controlled by hormones. Reciprocal blood transfusions between specimens of Cambarellus that had been on black and on white backgrounds for two hours revealed that the blood always contained pigmentdispersing and -concentrating hormones. The degree of pigment dispersion at any time appeared to be due to the relative quantity of each antagonist in the blood. Fingerman and Lowe (1957a) determined the effects of maintenance of specimens of Cambarellus on black and on white backgrounds for periods up to three weeks. The rates of red pigment dispersion and concentration in intact crayfish decreased progressively. In addition, red pigment gradually lost its inherent ability to concentrate after isolation. The titers of chromatophorotropins in the circumesophageal connectives of dwarf crayfish that had been on a black or a white background for two weeks changed significantly relative to the titers in crayfish that had been on the same shade of background for two hours (Fingerman and Lowe, 1957a). They found that the hormone not needed for proper adaptation to one of the backgrounds, e.g., red pigmentdispersing hormone of crayfish on a white background, was stored in the circumesophageal connectives. The quantities of chromatophorotropins in the blood also