Crustacean Hemolymph Research Articles

Synthesis of a high-density lipoprotein in the developing blue crab (Callinectes sapidus).

An important lipoprotein in the hemolymph of crustaceans is LpI. It transports lipid to peripheral tissues and also has a role in crustacean immune recognition. We employed a monoclonal antibody specific for the LpI peptide to demonstrate by ELISA, western blot and immunohistochemistry the appearance of LpI during development of Callinectes sapidus, the blue crab. LpI was first found in stage 5 embryos and appeared to be synthesized by lateral basophilic cuboidal cells that demonstrated cytoplasmic immunoreactivity for LpI at their interface with the yolk mass. The embryonic cuboidal cells bore a strong cytologic resemblance to the hepatopancreas cells of later stages (zoea, megalopae, adults), which were also immunoreactive for LpI.

The effects of serotonin and ecdysone on primary sensory neurons in crayfish.

The overall behaviors and motivational states observed during social interactions and throughout the molting cycle of crayfish have been linked to the effects of humoral neuromodulators. Both serotonin (5-HT) and a molt-related hormone, 20-hydroxyecdysone (20-HE), are known to be present in the hemolymph of crustaceans. To determine if they alter the activity of a primary sensory neuron that monitors proprioceptive information, we examined their effects on the activity of the slow-adapting muscle receptor organ (MRO) of the crayfish abdomen, a model sensory system that has been extensively studied. 5-HT within the range of 100 nM to 1 microM, increases the firing frequency of the neuron during sustained stimulation. In experiments in which 20-HE was added alone, an increase in the firing frequency also occurred, although to a lesser degree than that for 5-HT at the same concentrations. When the MRO is first exposed to 20-HE, followed sequentially by 5-HT, the activity increases to about the same degree as in the reverse order of exposure. This outcome indicates that mixtures of these endogenous neuromodulators, at various levels, are more important in alternating behavior than the absolute level of any one of them introduced alone.

Physiological and Biochemical Adaptations to Respiration of Hemoglobin-Containing Hydrobionts

Hemoglobin content in hemolymph of mollusks, crustaceans, and insect larvae, as well as in polychaete blood, is analyzed in the context of their respiratory conditions. The total supply of hydrobionts with respiratory pigment, as well as buffer capacity of hemolymph under normal and unfavorable conditions, is comparatively evaluated. The functions and properties of invertebrate hemoglobins are discussed.

Altered responsiveness to 5-HT at the crayfish neuromuscular junction due to chronic p-CPA and m-CPP treatment

Serotonin (5-HT) levels in the hemolymph of crustaceans has been implied to alter aggressiveness which influences social interactions. The activation of IP3 as a second messenger cascade within crayfish motor neurons in response to application of 5-HT, suggests that the 5-HT receptor subtypes on the motor neurons are analogous to the vertebrate 5-HT2A receptors. Based on evidence in other systems, it would be expected that chronically sustained 5-HT levels in aggressive individuals would result in a compensatory negative feed-back regulation and/or that target tissues would diminish their sensitivity to high levels of circulating, free 5-HT. We addressed the issue of up- and down-regulation in the sensitivity of the responsiveness to exogenously applied 5-HT at the NMJs of crayfish in which the animals have altered endogenous 5-HT levels. Injections of the 5-HT1 and 5-HT2 vertebrate receptor agonist, 1-(3-Chlorophenyl) piperazine dihydrochloride (m-CPP), for 1 week resulted in a decreased responsiveness to application of 5-HT. The compound p-chlorophenylalanine (p-CPA) blocks the enzymatic synthesis of 5-HT and following 7 days of p-CPA injections, a super-sensitivity to exogenous application of 5-HT for both tonic and phasic neuromuscular junctions (NMJs) was observed. However, acute applications of p-CPA and m-CPP, followed by extensive saline washing, did not reveal any altered receptivity to 5-HT application.

Isolation of lectins from hemolymph of decapod crustaceans by adsorption on formalinized erythrocytes

Hemolymph of decapod crustaceans contains lectins of important specificity. An isolation procedure, based on adsorption of hemolymph lectins on red blood cells (RBC) fixed with formaldehyde, is described. Hemolymph is let to clot for 3 h at 22–28°C (RT) and for 24 h at 5°C; centrifuged at 13 000 g for 30 min; filtered through 5-μm filters; diluted with an equal volume of 50 mM NaCl, 100 mM CaCl 2; supplemented with protease as well as phenoloxidase inhibitors; centrifuged at 13 000 g for 20 min. Formalinized RBC (FRBC) are mixed with diluted hemolymph to a suspension of about 20% v/v FRBC. After incubation for 30 min at RT, FRBC are washed five times with 150 mM NaCl, 10 mM CaCl 2. The lectins adsorbed on FRBC are desorbed using either 100–500 mM of carbohydrate solutions in 0.9% NaCl or 50 mM Tris–HCl buffer, pH 8.0 containing 100 mM NaCl and 20 mM entylenediaminetetraacetate (EDTA). The procedure is efficient in isolating the hemolymph lectins of the decapods Liocarcinus depurator and Potamon potamios.

The divers problems associated with sampling crustacean haemolymph in the wild west (Wales/Indies).

The divers problems associated with sampling crustacean haemolymph in the wild west (Wales/Indies).

In vitro Nodule Formation in the Ridgeback Prawn, Sicyonia ingentis, and the American Lobster, Homarus americanus

Bacteria injected into the hemolymph of crustaceans are rapidly cleared from circulation; many accumulate in the gills, even though this organ does not contain fixed phagocytic cells. In this study, hemolymph from a shrimp or a lobster was mixed in a culture medium with or without bacteria. In all cases, hemocytes rapidly aggregated to form nodules. As nodules increased in size, the number of free hemocytes, nodules, and bacteria (if present) decreased. Nodule formation was inhibited by the addition of the peptide RGD (Arg-Gly-Asp). The morphology of the nodules formed in the presence and absence of bacteria was examined using light and electron microscopy. Within 10 min, nodules were larger than the small blood spaces of the gills, suggesting that in vivo clearance is the result of physical entrapment of nodules and foreign material in the narrow vessels of the gills. This method of removing foreign material from circulation is discussed in relation to other clearing mechanisms in crustaceans. Additional key words: Crustacea, hemocytes, immunity, gills Bacteria or particles of similar size injected into the hemolymph of decapod crustaceans are rapidly removed from circulation (Cornick & Stewart 1968; McKay & Jenkins 1970; Merrill et al. 1979; White & Ratcliffe 1982). When one million bacteria are injected into a 15-20 g (wet weight) shrimp, Sicyonia ingentis, with an estimated hemolymph volume of 4-6 ml, 80100% of the bacteria are eliminated within 5 min (Martin et al. 1993). Studies with radioactive tracers and dyes show that the gills are the major sites of clearance in shrimps (Martin et al. 1993) and in other decapods, and that they share this role with other organs, in particular the digestive gland (McCumber & Clem 1977; Merril et al. 1979; White & Ratcliffe 1982; Mullainadhan et al. 1984). In most decapods, fixed phagocytic cells lining the vasculature of the digestive gland ingest the foreign material (Johnson 1987; Factor & Beekman 1990; Factor & Naar 1990; Sagrista & Durfort 1990; Factor 1995), but these cells are absent from the digestive gland of shrimps (Johnson 1987; Martin et al. 1993). The mechanism by which foreign materials are cleared from circulation and accumulated in the gills of decapods is unknown. Although cells capable of pinocytosis have been demonstrated in the gills a Author for correspondence. E-mail: gmartin@oxy.edu (Strangways-Dixon & Smith 1970; Foster & Howse 1978; Johnson 1987; Doughtie & Rao 1981), no population of phagocytic cells has been found. Several studies have shown that the number of circulating hemocytes drops significantly following the injection of foreign materials into the blood (Cornick & Stewart 1968; Tyson & Jenkins 1973; Paterson et al. 1976; Smith & Soderhall 1983; Smith et al. 1984; Martin et al. 1993) and that aggregations of hemocytes subsequently occlude the vasculature of the gills (Smith & Ratcliffe 1980; White et al. 1985). Crustacean hemocytes, in addition to containing phenoloxidase, which may initiate immune responses (Soderhall 1982; Johansson & Soderhall 1996), also contain a cell adhesion protein (Johansson & Soderhall 1992) that may serve to bind hemocytes and foreign material in tight aggregations called nodules. If these nodules become sufficiently large, they may become physically trapped in narrow vessels of the body, particularly in the gill vasculature. In this study, we describe nodule formation in vitro with particular attention to the rate of nodule formation, the size of the nodules, and the ability of the nodules to clear bacteria from suspension. In addition, we were interested if the peptide RGD (Arg-Gly-Asp), which is important in cell adhesion studies (Akiyama & Yamada 1985; Hayman et al. 1985), could inhibit This content downloaded from 207.46.13.159 on Sat, 22 Oct 2016 04:37:41 UTC All use subject to http://about.jstor.org/terms Martin, Kay, Poole, & Poole nodule formation in vitro. These data, when compared to diameters of gill-vessels in the gill, support the suggestion that enlarging nodules are physically trapped in the fine vasculature of the gills, where the foreign material may then be degraded by hemocytes.

The clearance in vivo and metabolism of ecdysone and 3-dehydroecdysone in tissues of the crabCancer antennarius

The Y-organs of Cancer antennarius secrete the hormones ecdysone (E) and much greater amounts of 3-dehydroecdysone (3DE), but the dominant derivative in the circulation is 20-hydroxyecdysone (20E). The uptake and clearance of E, 3DE, and metabolites were compared in selected tissues in vivo, and the capacity of each to generate 20E was assessed in vitro. [3H]-labeled E or 3DE, injected into intermolt crabs, rapidly cleared from hemolymph, while being taken up and then rapidly cleared from tissues (muscle, heart, epidermis, ovary) within 1 h. Labeled 20E from E or 3DE appeared in all tissues in the first hour, cleared to varying degrees from tissues, and then reaccumulated over 24 h. Among these tissues, ovary accumulated the most labeled 20E. Generally by 24 h more labeled 20E accumulated from 3DE than from E. Excising the eyestalks (inducing premolt) did not affect the outcomes except that deeyestalking caused much greater concentrations of 3DE than E to appear in all tissues initially. Tissue extracellular volumes ([14C]inulin spaces) were not altered by deeyestalking, except transiently in epidermis, and otherwise did not account for the observed ecdysteroid dynamics. A parallel study with eyestalk neural tissue from crabs receiving [3H]3DE showed extraordinary accumulation of 3DE and labeled metabolites. The normalized tissue/hemolymph ratio was 72 at 8 h, compared with 0.6–2.6 for other tissues. The apparent extracellular volume of eyes was high (80%) but did not account for the high ecdysteroid accumulation. Tissues were incubated 18 h with [3H]3DE. Activities representing 3β-reductase and 20-hydroxylase generally were present, evidenced by finding labeled E and 20E. Tissue blanks, hemolymph, and muscle were devoid of activity, hepatopancreas and epidermis were intermediate, ovary, testis, and hindgut were high, and eyestalk tissue was highest in activities. Low amounts of labeled high-polar material were usually present, and a labeled derivative tentatively identified as 3-dehydro-20-hydroxyecdysone (3D20E) also appeared in ovary, hepatopancreas, hindgut, and eyes, indicating some direct conversion of 3DE to 3D20E. In composite, these data indicate that 3DE secreted by Y-organs is converted to E, thence to 20E by several tissues, accounting for the preponderance of 20E in crustacean hemolymph. The findings support the hypotheses that 20E is the definitive molting hormone (receptor ligand) and that an ecdysteroid feedback mechanism controls release of the molt-inhibiting hormone from the eyestalks. J. Exp. Zool. 279:609–619, 1997. © 1997 Wiley-Liss, Inc.

Mesanophrys chesapeakensis n. sp., a Histophagous Ciliate in the Blue Crab, Callinectes sapidus, and Associated Histopathology

Histophagous ciliate infections were infrequently detected in blue crabs, Callinectes sapidus, from Chesapeake Bay, Delaware Bay, and a coastal bay of Maryland. Associated pathology included hemocyte aggregations in various tissues and nodule formation. Protargol silver staining of cultured ciliates revealed an oral apparatus morphologically similar to that of other hemolymph-infecting ciliates of the genus Mesanophrys. The oral apparatus included three distinct oral polykinetids and an oral dikinetid consisting predominantly of a b segment and a smaller c segment. Morphologic comparison to other histophagous ciliates infecting crustaceans showed that the ciliate in blue crabs is a distinct species, Mesanophrys chesapeakensis n. sp., family Orchitophryidae. Additional key words: protargol, Orchitophryidae Histophagous ciliates from wild and captive crustaceans have been reported sporadically from various locations since 1888. The ciliate Mesanophrys (Anophrys COHN 1866) maggii CATTANEO 1888 was first reported parasitizing the hemolymph of a European shore crab, Carcinus maenus LINNAEUS 1758, from Italy. Incomplete or poor morphological descriptions of several crustacean-infecting ciliates have caused considerable confusion and elicited much discussion on systematics (Armstrong et al. 1981). Based on the morphology of oral structures of protargol-stained specimens, a new genus, Mesanophrys, was erected by Small & Lynn (1985) to include ciliates with only a short oral dikinetid and other features now found in the genera of the family Orchitophryidae. Two species, Mesanophrys (Paranophrys) maggii CATTANEO 1888, and Mesanophrys (Anophrys) carcini GROLIERE & LEGLISE 1977 (Small & Lynn 1985), were transferred into this newly erected genus; a third species, Mesanophrys pugettensis MORADO & SMALL 1994, was recently reported. However, incorrect generic placements of histophagous ciliates from crustaceans have continued, as discussed by Morado & Small (1994). Recently, the family Paranophryidae (JANOWSKI, in Small & Lynn 1985) was determined to be a junior synonym a To whom correspondence should be addressed: Gretchen Messick 410-226-5193 FAX: 410-226-5925 e-mail gmessick @ hatteras.bea.nmfs.gov. of Orchitophryidae CEPEDE 1907, due to rule of priority (Morado & Small 1994). Orchitophrya stellarum CEPEDE 1907 was recently reinvestigated using protargol staining and was determined to be a scuticociliate rather than an astome (Bouland et al. 1986). Thus, Paranophryidae falls as a junior synonym to Orchitophryidae (Morado & Small 1994). Most reports of ciliates in crustacean hemolymph involve captive hosts (Cattaneo 1888; Bang et al. 1972; Aiken et al. 1973; Groliere & Leglise 1977; Armstrong et al. 1981; Sparks et al. 1982). Reports of histophagous ciliates in wild crustacean populations are rare, and infection prevalence has typically been low. Poisson (1930) found only 0.2% (7 of 3000) shore crabs, C. maenus, infected with histophagous ciliates. Infections in wild stocks of the American lobster, Homarus americanus H. MILNE EDWARDS 1837, were found only three times in 14 years (Aiken & Waddy 1986). In contrast to these reported low prevalence levels in hemolymph of wild lobsters, a ciliate was present in smears of hepatopancreas tissue of all freshly caught Maine lobsters, H. americanus (n=89), although the ciliate was often undetected in hemolymph preparations (Sherburne & Bean 1991). Mesanophrys pugettensis was found in fewer than 0.4% (n=1916) of wild and captive Dungeness crabs, Cancer magister DANA 1852, although it was reported in 3% (n=87) of wild rock crabs, C. productus RANDALL 1839, and 29% (n 7) of wild kelp crabs, Pugettia producta RANDALL This content downloaded from 157.55.39.153 on Mon, 19 Sep 2016 04:45:06 UTC All use subject to http://about.jstor.org/terms

Cation balance in crustacean haemolymph: relationship to cell membrane potentials and membrane surface charge

Data were compiled on K+, Na+, Mg2+ and Ca2+ in haemolymph of 74 species of Malacostraca and three freshwater species of Anostraca. Mean [K+] is positively correlated with both mean [Na+] and mean [Mg2+]. Mean [Mg2+] and [Na+] are themselves positively correlated. Mean [Ca2+] shows little correlation with the others, except that concentrations of all four cations are low in the Anostraca. The data suggest that haemolymph composition has evolved in such a way as to preserve the transmembrane potential across particular areas of cell membrane, this being determined by constant transport and permeability properties for K+, Na+ and Ca2+, and by superficial fixed negative charges of constant density (about 1 e/nm2 on the outer surface).

Cell-Free Synthesis of Vitellin in the Shrimp Penaeus semisulcatus (de Haan)

The vitellogenic ovary of Penaeus semisulcatus contains mRNA specific for vitellin (Vt), low levels of which are found in nonvitellogenic ovaries, and is absent from testes. lmmunoisolation from cell-free translation of poly(A) + RNA using antiserum against purified Vt produced a 35-kDa polypeptide. No differences were found between Vt precipitated from the translation products of the rabbit reticulocyte system and that from the wheat germ extract. The specificity of the immunoprecipitation reaction was demonstrated by the absence of precipitation with nonimmunized rabbit serum and with antibodies prepared against purified hemocyanin or lipoprotein I (LPI), which are crustacean hemolymph proteins. In addition, competition with the radioactively labeled translation product occurred only in the presence of purified Vt, but not BSA or LPI. Vt synthesized in the translation system had a significantly lower molecular weight than that of the purified Vt or that synthesized by ovaries incubated in vitro. The possibility that this difference may be because of the nonglycosylated nature of a cell-free translation product was tested. The removal of oligosaccharides from purified Vt by enzymatic digestion with N-glycosidase F resulted in the appearance of a smaller polypeptide of 36 kDa, which reacted immunologically with Vt antiserum in a Western blot. The size of this fragment is very close to the molecular weight of the translation product.

Comparison of hemolymph lipoproteins from four species of crustacea

AbstractTwo different lipoproteins, lipoprotein I and very high density lipoprotein (VHDL), were isolated from the male hemolymphs of freshwater prawn, mitten crab, and striped stone crab by a different density gradient ultracentrifugation. Lipoprotein II as well as lipoprotein I and VHDL was further found in the female hemolymphs of these crustacea. Lipoprotein II seemed to be a vitellogenin and spawned kuruma prawn without eggs lacked lipoprotein II in their hemolymph. The densities of lipoprotein I varied between 1.12 and 1.18 g/ml reflecting the difference of protein‐lipid ratio. In contrast lipoprotein II and VHDL had approximately constant densities of 1.19—1.21 g/ml and 1.26—1.27 g/ml, respectively. Lipoprotein I, lipoprotein II, and VHDL isolated from the crustacean hemolymph contained phospholipid as a predominant lipid component. Crustacean lipoprotein was divided into the freshwater type and the seawater type. Freshwater prawn and mitten crab classified into the freshwater type tended to have high protein concentrations in lipoprotein I and VHDL. In contrast both lipoprotein I and VHDL protein concentrations from kuruma prawn and striped stone crab, which were classified into the seawater type, were much lower than those from the freshwater type. Lipoprotein I from freshwater prawn and mitten crab had two major apolipoproteins with apparent molecular weights of 230,000 and 100,000, and 340,000 and 100,000, respectively, while lipoprotein I from kuruma prawn and striped stone crab consisted of three major apolipoproteins (molecular weights 180,000, 100,000, and 80,000). VHDL found for the first time in crustacea probably plays an important role in lipid transfer reaction between lipoproteins. © 1993 Wiley‐Liss, Inc.

Quantification of methyl farnesoate levels in hemolymph by high-performance liquid chromatography

Methyl farnesoate (MF) is an acyclic sesquiterpenoid that has been detected in hemolymph and other tissues of crustaceans and insects. This paper describes a rapid and sensitive method for measuring MF in crustacean hemolymph. Extracts of hemolymph samples were separated by normal-phase high-performance liquid chromatography (5-μm silica, 250 × 4.6 mm I.D., 1.3% diethyl ether in hexane) and detected by UV (220 nm). The limit of detection with this method was less than 250 pg/ml. This method should be useful for studying the physiological functions of MF in crustaceans and other arthropods.

Photoaffinity labeling and biochemical characterization of binding proteins for pheromones, juvenile hormones, and peptides

Radiolabeled photoaffinity analogs can be used to purify and characterize proteins involved in pheromone perception, juvenile hormone (JH) action, and neuropeptide reception. Several photoaffinity analogs and purification strategies are described for each of these physiological targets. First, a diazoacetate photoaffinity label is used to selectively modify the pheromone binding protein of the gypsy moth, Lymantria dispar. Reverse-phase HPLC is then employed to fractionate the male antennal proteins. Second, a tandem procedure involving preparative isoelectric focusing (IEF) and ion-exchange (IEX) HPLC is employed for the purification of the Manduca sexta hemolymph juvenile hormone binding protein (JHBP), which has now been cloned and sequenced. A separate application of this strategy for the purification of the 29 kDa JH I/methoprene receptor proteins from epidermal nuclei of M. sexta larvae is outlined. A new photolabel, farnesyl diazoketone, has been employed for the characterization of crustacean hemolymph methyl farnesoate binding proteins. Third, the development of neuropeptide photoaffinity labels is described for two systems: (1) the red pigment concentrating hormone (RPCH) of shrimp and (2) the allatostatins isolated from the brain of the cockroach Diploptera punctata.

Lipoproteins I and II from the hemolymph of the blue crab Callinectes sapidus: Lipoportein II associated with vitellogenesis

AbstractTwo different high density lipoproteins were isolated and characterized in the hemolymph of blue crabs (Callinectes sapidus). Lipoprotein I was found in both sexes and comprised one peptide (112,000 daltons) and lipid, primarily phosphtidyl choline. Lipoprotein I properties included a hydrated density of 1.12 g/ml, sedimentation coefficient of 5.4 Svedberg flotation units and a protein to lipid ratio of 1.0. This lipoprotein is assumed to have a role in transporting lipid from the hepatopancreas to other tissues. Lipoprotein II had three peptides (190,000, 107,000 and 78,000 daltons), phosphatidyl choline as the major lipid, a hydrated density of 1.16 g/ml, a sedimentation coefficient of 10.4 Svedberg flotation units, a protein to lipid ratio of 1.1, and a yellow to orange color due to a mixture of carotenoids. Lipoprotein II was found only in female crabs undergoing vitellogenesis. Lipoprotein II appeared after 30 days in females that had mated and completed their final molt. The maximum concentration was reached in 50 to 60 days. Lipoprotein II was not present in recently molted females in a collection of crabs from one esturine river but was present in all other stages with highest concentrations (up to 8 mg/ml hemolymph) in stages 5 and 6 (females with large, well developed ovaries). Crab ovaries had a lipoprotein similar to hemolymph Lipoprotein II. Lipoprotein II probably plays an important role in carrying lipid and specific peptides to ovarian tissues. Crustacean hemolymph lipoproteins show some similarities to insect lipophorins, but are quite different from vertebrate lipoproteins.

Acid-base balance and blood gas changes in the fresh water field crab, Barytelphusa guerini, on exposure to organic and inorganic lead.

The acid-base status of crustacean haemolymph depends on various environmental and physiological factors. Acid base status of the haemolymph is known to be influenced by temperature, salinity, strenuous activity and moulting. The studies on the acid-base regulation of the fresh water crabs are meager. The acid-base changes in fishes during environmental stress conditions like acid stress and zinc toxicity had been reported. But the effect of environmental pollutants like the heavy metals on the acid-base regulation of the fresh water crabs have not been previously reported. The haemolymph of the fresh water crab was found to accumulate high amounts of lead on exposure to organic and inorganic lead. Hence the present investigation has been undertaken to study the haemolymph acid-base status on exposure to subtoxic levels of organic and inorganic lead.

Ionic regulation in crustacea: The influence of temperature on apparent set points

1. 1. Homeostatic set points have usually been neglected in discussions of temperature effects on ionic regulation. 2. 2. Using published data for crustacean haemolymph on Na +, Cl −, freezing point depression and conductivity, this paper explores the effects of temperature on iso-ionic and isosmotic concentrations and on concentrations representing particularly effective hyper-regulation. 3. 3. It is argued that these concentrations approximate to set points, though the exact nature of the regulated variable(s) is unclear. 4. 4. As temperature increases most of these concentrations stay constant or fall slightly; none rises or falls by more than 2%/°C. 5. 5. The few available data on K +, Ca 2+ and Mg 2+ are considered briefly.

A new operational apporach to P CO 2 determination in crustacean hemolymph

A new method for calculating P CO 2 based on mathematical expressions derived from a measured ‘Davenport diagram’ is decribed. The measured ‘Davenport diagram’ is constructed from in vitro buffer curves relating total CO 2 to pH at P CO 2 levels which adquately encompass the in vivo range of the acid-base status for the species in question. The ‘Davenport diagram’ is described by three linear equations such that P CO 2 can be accurately calculated from in vivo measured C CO 2 and pH. The equations are specific for a given species at a given temperature and hemolymph ionic strength, as are the constants in the more classical Henderson-Hasselbaclh equation. The accuracy of the method is equal to calculation using the Henderson-Hasselbalch equation with corrected values for p K 1 ′ αCO 2 and in vivo P CO 2 measured directly. This procedure is equally applicable to fluids with dissolved pigments such as hemolymph from freshwater and marine crustaceans and to human blood. The major benefit pf this method of calculating P CO 2 is that correction nomograms for the constants in the Hendersen-Hasselbalch equation are not required.

Comparative biochemical studies on plasma cholesterol. II. Relationship between plasma esterified cholesterol and lecithin: Cholesterol acyltransferase activity.

In five animals, blue crab, crayfish, carp, rat and man; their plasma lecithin: cholesterol acyltransferase (LCAT) activities were determined from the aspect of comparative biochemistry. The activities of mammalian blood plasma are much higher than those of carp blood and crustacean haemolymph plasma. Moreover, it was found that there is an apparent linear relationship between the LCAT activities and the ratios of cholesterol ester to total cholesterol. But the ratios of triglycerides to total lipids do not correlate with the LCAT activities, differing from the case of the diabetic mellitus patients. Therefore, it is probable that the plasma esterifying rate of cholesterol is governed by the ratios of cholesterol ester/total cholesterol, and it seems that plasma esterofied cholesterol increases with the evolution of animals.

Uptake of the yolk protein, lipovitellin, by developing crustacean oocytes

A variety of cytochemical techniques were used to demonstrate how crustacean lipovitellin accumulates within the egg. It was found that a protein serologically identical to the lipovitellin of yolk spheres was present in the hemolymph of vitellogenic crustaceans, but was absent from the hemolymph of males and immature females. In the three crustacean species studied ( Uca pugilator, Cambarus clarkii, and Libinia emarginata), pinocytosis of fluorescein-conjugated lipovitellin and trypan blue occurred only during those periods when oocytes were accumulating yolk. It may be concluded from the present studies that yolk spheres develop in crustacean eggs primarily through micropinocytotic uptake of lipovitellin from the hemolymph, although other oocyte proteins appear to be made in the oocyte.