Haemolymph Of Periplaneta Americana Research Articles

Characterization of a tyrosine phosphatase activity in the oogenesis ofPeriplaneta americana

In this work, phosphatase activity was characterized in the ovary and the haemolymph of Periplaneta americana. The optimum pH for these activities was 4.0, and a temperature of 44 degrees C was ideal for the maximal enzyme activity. The phosphatase activities were inhibited by NaF, sodium tartrate, Pi, sodium orthovanadate, and ammonium molybdate. The ovarian phosphatase activity at pH 4.0 was almost exclusive against phosphotyrosine, with little or no effect on the residues of phosphoserine or phosphothreonine. These results indicate that this phosphatase activity is due to the presence of an acid tyrosine phosphatase. The phosphatase activities of acid extracts from P. americana ovaries (OEX) and an acid extract from P. americana haemolymph (HEX) were analyzed in non-denaturant gel electrophoresis using an analog substrate beta-naphtyl phosphate. The gel revealed two bands with phosphatase activity in the ovary and one band in the haemolymph; these bands were excised and submitted to a 10% SDS-PAGE showing a single 70-kDa polypeptide in both samples. Histochemistry of the ovary with alpha-naphtyl phosphate for localization of acid phosphatase activity showed mainly labeling associated to the oocyte peripheral vesicles, basal lamina, and between follicle cells. Electron microscopy analysis showed that acid phosphatase was localized in small peripheral vesicles in the oocyte, but not inside yolk granules. The possible role of this phosphatase during oogenesis and embryogenesis is also discussed in this article.

Molecular cloning of cDNA for lipopolysaccharide-binding protein from the hemolymph of the American cockroach, Periplaneta americana. Similarity of the protein with animal lectins and its acute phase expression

A previous paper described the purification of a calcium-dependent lipopolysaccharide-binding protein from the hemolymph of Periplaneta americana (Jomori, T., Kubo, T., and Natori, S. (1990) Eur. J. Biochem. 190, 201-206). This paper describes the molecular cloning and characterization of cDNA for the LPS-binding protein. This protein was found to have a carbohydrate-recognition domain at its carboxyl terminus containing amino acid sequences that are conserved in various mammalian C-type lectins. It was also shown to contain an N-linked carbohydrate chain, and the amino acid residue carrying this chain was assigned as Asn at position 56 (23rd amino acid residue from the amino terminus). Northern blot analysis revealed the presence of multiple mRNAs that hybridized with this cDNA and transient increases in their content after injection of Escherichia coli into adult Periplaneta, suggesting that the LPS-binding protein plays a role in the acute phase response of this insect.

Catecholamines and related o-diphenols in cockroach hemolymph and cuticle during sclerotization and melanization: comparative studies on the order Dictyoptera

Catecholamines and related o-diphenols extracted from the cuticle and hemolymph of adult cockroaches during sclerotization and pigmentation of the cuticle were analyzed by reverse phase HPLC with electrochemical detection. At ecdysis, dopamine (DA) o-conjugates predominated in the hemolymph of Periplaneta americana, P. australasiae, P. fuliginosa, P. brunnea, and Blatta orientalis (Blattidae); Blattella germanica (Blattellidae); and Gromphadorhina portentosa and Blaberus craniifer (Blaberidae). N-Acetyldopamine (NADA) conjugates were second in abundance in these species, but were major in the hemolymph of the other blaberoid species, Leucophaea maderae and Nauphoeta cinerea. After ecdysis NADA became the major hemolymph catecholamine in all species as DA decreased rapidly. N-beta-Alanyldopamine (NBAD) concentrations in the hemolymph remained low in all species, although NBAD and its metabolite, N-beta-alanylnorepinephrine (NBANE), were generally the major catecholamines in tanning cuticle. Catechol (1,2-dihydroxybenzene) occurred mainly as a conjugate(s) at high levels in the hemolymph of nymphs and adults of all blattid species. Only trace amounts were detected in B. germanica and Cryptocercus punctulatus (Cryptocercidae), and none was found in any of the blaberoid species. High concentrations of NBANE and NBAD accumulated in tanning cuticle of B. germanica, G. portentosa, and all blattid species, whereas NADA and DA predominated in cuticle from the other blaberoid species, particularly L. maderae and N. cinerea. However, cockroaches as a group appear to utilize both the N-acetyl and N-beta-alanyl catecholamines for stabilization of the exoskeleton. The Blattidae differed most from the other families in having considerably higher concentrations of catecholamines in hemolymph and cuticle, as well as the large amounts of catechol conjugates in the hemolymph.

Purification and characterization of lipopolysaccharide‐binding protein from hemolymph of American cockroach Periplaneta americana

A protein having affinity to lipopolysaccharide of Escherichia coli K12 was purified to homogeneity from the hemolymph of Periplaneta americana. This protein, with an average molecular mass of 450 kDa. was a homooligomer of a 28-kDa subunit protein. Comparative studies using lipopolysaccharide molecules of E. coli and Salmonella minnesota suggested that this protein recognizes and binds to a specific carbohydrate structure of E. coli lipopolysaccharide. Ca2+ was required for this protein to bind to lipopolysaccharide, but other divalent cations could not replace Ca2+.

Diphenols in hemolymph and cuticle during development and cuticle tanning of Periplaneta americana (L.) and other cockroach species

AbstractDiphenolic compounds in cockroach hemolymph and cuticle were extracted with 1.2 N HCI, partially purified by alumina adsorption, and analyzed by liquid chromatography. Dopamine (DA) is the major catecholamine in hemolymph of Periplaneta americana, Blatta orientalis, Blattella germanica, Gromphadorhina portentosa, and Blaberus craniifer at adult ecdysis, while N‐acetyldopamine (NADA) predominates in hemolymph of Leucophaea maderae. In P. americana, NADA is the second most abundant catecholamine, while N‐β‐alanyldopamine (NBAD), norepinephrine (NE), 3,4‐dihydroxyphenylalanine, 3,4‐dihydroxyphenylethanol, 3,4‐dihydroxyphenylacetic acid, and 3,4‐dihydroxybenzoic acid occur in lesser quantities. Catecholamines occur mainly as acid labile conjugates in hemolymph. Dopamine, conjugated primarily as the 3‐sulfate ester, increases in hemolymph from 0.1 to 0.8 mM during the last instar. Concentrations decrease by 75% in pharate adults, partially because of an increase in hemolymph volume. A second smaller peak of DA sulfate occurs after ecdysis followed by a rapid disappearance as the cuticle tans. A conjugate of catechol (o‐dihydroxybenzene) is also present in relatively high concentrations at all ages examined. In cuticle, N‐β‐alanylnorepinephrine accumulates during the early period of adult tanning, while NBAD, NADA, N‐acetylnorepinephrine, and DA increase more slowly. The N‐β‐alanyl and N‐acetyl derivatives of DA and NE occure in relatively high concentrations in tanned cutical of P. americana and probably play an important role in the stablization process.

Purification and some properties of a lectin from the hemolymph of Periplaneta americana (American cockroach).

A lectin showing specificity for human A-type red blood cells was purified to homogeneity from the hemolymph of the American cockroach Periplaneta americana by affinity chromatography on bovine submaxillary gland mucin. This lectin was a huge molecule with molecular mass of about 1500 kDa, with a single subunit of 30-kDa protein, and required Ca2+ for expression of lectin activity. Electron microscopic examination showed that these molecules were rods with helical structure with an average length of 50.5 nm and width of 10 nm. The molecule was suggested to contain tandemly aligned basic units of 10 nm length.

High molecular transport proteins for JH-III in insect hemolymph

Using two independent techniques, ultracentrifugation in a KBr-gradient, and native pore polyacrylamide gel electrophoresis in combination with [3H]-epoxyfarnesyldiazoacetate photoaffinity labeling, we showed that in the hemolymph ofPeriplaneta americana, and probably also inLeptinotarsa decemlineata JH-III binds to the lipophorin, whereas inLocusta migratoria JH-III binds to a different protein.

Activation, properties and collection of haemolymph phenoloxidase of the american cockroach, Periplaneta americana

1. The haemolymph of Periplaneta americana contains the proenzyme prophenoloxidase (EC 1.14.18.1) which was activated by incubation of the haemolymph with various organic solvents. Their effectiveness was acetone > methanoi > ethanol > propanol > butanol. 2. Activated phenoloxidase oxidized diphenols, d- and l-Dopa, pyrocatechol, epinephrine and norepinephone and the triphenol pyrogalcol, but showed no activity towards tyrosine or other monophenols. 3. Various methods of drawing haemolymph were evaluated for their effectiveness in recovering activatable phenoloxidase activity. Acetic acid pretreatment of the whole insect prior to collecting haemolymph was found to be the best, apparently due to its effectiveness in inhibiting haemocyte coagulation. 4. The activated enzyme was inhibited most effectively by phenylthiourea and had a pH optimum of 6.

Further characteristics of adipokinetic and hyperglycaemic factor(s) of stick insects

When an extract of the corpora cardiaca/corpora allata from two species of wingless stick insects, Carausius morosus and Cuniculina impigra, which cause no adipokinetic or hyperglycaemic effect when injected into the donor insects themselves, is injected into adult Locusta migratoria it resulted in an increase in the haemolymph lipid concentration. The lipid elevation was time dependent, with a maximum effect about 90–180 min after injection, and was also dose-dependent. About 0.001–0.002 ( C. morosus) and 0.01 ( C. impigra) gland equivalents were needed to produce a significant increase; a maximal effect was reached with approx. 0.075 ( C. morosus) and 0.25 ( C. impigra) gland equivalents. Carausius extract was also able to elevate carbohydrate concentration in the haemolymph of Periplaneta americana. However, the effect was weak and no maximal response was reached even with a dose of 0.5 gland equivalents. Adipokinetic hormone activity was present in CC/CA extracts of larval Carausius; the activity was about 30 times lower in 1-day-old 2nd instar individuals, and approx. 5 times less at the beginning of the 6th instar than that found in adults. In both stages the hormone levels increased gradually from the beginning to the end of the instar. No age-related changes were observed during the adult stage. Further studies on the lipid-mobilising factor of C. morosus revealed that it was stored entirely in the CC and not in other nervous tissue, e.g. brain, CA, suboesophageal ganglion, thoracic and abdominal cord. The factor was heat stable for at least 1 hr at 100°C and retained its adipokinetic activity after incubation with trypsin and the exopeptidases such as carboxypeptidase A and leucine aminopeptidase. However, activity was abolished when incubated with thermolysin and α-chymotrypsin. From these experiments a close resemblance to the locust AKH, a blocked decapeptide, is suggested.

A study of ion binding in the hemolymph of Periplaneta americana

1. 1. The concentrations of the major inorganic ions in the hemolymph of Periplaneta americana were determined as follows (in mM): Na +, 132; K +, 8.7; Mg 2+, 4.7; Ca 2+, 3.7; Cl −, 107; PO 3− 4, 1.04. 2. 2. By the method of ultrafiltration, significant binding to macromolecules in hemolymph was demonstrated for the following ions: Na +, 22.0%; Mg 2+, 25.5%; Ca 2+, 16.2%; C1 −, 10.3%; PO 3− 4, 26.9%. 3. 3. K + was not bound significantly to macromolecules in hemolymph.

In vitro biosynthesis of oöthecal sclerotization agents from tyrosine by haemolymph of Periplaneta Americana

Whole haemolymph of the adult female American cockroach was homogenized in distilled water and the extract subjected to column chromatography on Sepharose-6B. The partially purified enzyme, when incubated with the appropriate cofactors, was capable of catalyzing several reactions. With pyridoxal-phosphate and NAD but without the addition of copper, l- 14C(U)-tyrosine was metabolized to tyramine, octopamine, p- hydroxymandelic acid, p- hydroxybenzaldehyde , p- hydroxybenzylalcohol , p- hydroxybenzoic acid, p- hydroxyphenylpyruvic acid, p- hydroxyphenylacetic acid, p- hydroxyphenyllactic acid, and p- hydroxyphenylpropionic acid. The major metabolites of l-2′- 14C-tyramine were octopamine, p- hydroxymandelic acid and p- hydroxybenzoic acid. Further experiments using the crude homogenate and unlabeled substrates showed that octopamine was metabolized to p- hydroxymandelic acid and that it produced p- hydroxybenzaldehyde , p- hydrosybenzoic acid, and 3,4-dihydroxybenzoic acid (protocatechuic acid). These results suggest the following pathway exists for the synthesis of protocatechuic acid: tyrosine → tyramine → octopamine → p- hydroxymandelic acid → p- hydroxyphenylglyoxylic acid and/or p- hydroxybenzylalcohol → p- hydroxybenzaldehyde → p- hydroxybenzoic acid→protocatechuic acid. Hydroxylation of the p- hydroxy compounds occurred in the presence of copper but at different rates. The initial transamination and/or deamination of tyrosine to the p- hydroxypyruvate is apparently not instrumental in the synthesis of protocatechuic acid although the results of this investigation do not offer conclusive evidence concerning this question.

Vertebrate serum inhibitors of Aedes aegypti trypsin

A trypsin from the midgut of Aedes aegypti was partially purified. Its molecular weight was estimated as 21,500. This enzyme hydrolysed α-N- benzoyl- L-arginine ethyl ester (BAEE) and 2-N- benzoyl- DL-arginine -p- nitroanilide HCl (BAPNA). For the trypsin esterolytic activity the pH optimum was 7.9 and Michaelis-Menten constants were 0.47 m M for BAPNA and 2.24 m M for denatured bovine haemoglobin. The trypsin esterolytic activity was inhibited by the sera of 17 vertebrate species and by the haemolymph of Periplaneta americana. The inhibition capacity was relatively high in birds and low in an elasmobranch, with mammals, a reptile, a frog, teleosts, and an insect in the middle range. The number of inhibitors and their approximate molecular weights in each serum was studied by Sephadex gel chromatography.

Changes in the diuretic and antidiuretic properties of the hemolymph during the six-day vitellogenic cycle in the American cockroach

The “antidiuretic principle” concentration in the hemolymph of Periplaneta americana (L.) is low during the initial period of the six-day vitellogenic cycle. On the fourth day the concentration begins to rise and reaches a maximum on the fifth and sixth days of the cycle. The diuretic factor remains relatively constant except on day three when a peak is seen. However, the increase in “antidiuretic principle” on days 4,5, and 6 may mask a similar rise of diuretic activity. It is postulated that the physiological role of the antidiuretic principle is in conservation of water to counteract the tremendous water loss via the eggs. However, the possibility that the principle is actually a smooth-muscle stimulating hormone which enhances the movement of the eggs down the oviduct cannot be ruled out. There may be two components involved with each one having overlapping functions.

Fatty acid composition of the glycerine and free fatty acid fractions of the fat body, and hemolymph of the cockroach, Periplaneta americana (L.)

1. 1. The concentration and fatty acid composition of the tri-, di- and monoglycerides and free fatty acids of the fat body and hemolymph of Periplaneta americana (L.) were determined by column and gas-liquid chromatoraphy. 2. 2. Triglycerides increased in the fat body and decreased in the hemolymph of eight-instar roaches. In the young adults, triglycerides decreased markedly in the fat body and hemolymph; diglycerides increased in the hemolymph. 3. 3. The changes in the concentrations of fatty acids with chain lenghts of 16 and 18:1 with age were inversely related. 4. 4. The changes with age of the percentage of saturated fatty acids in the triglycerides and free fatty acids suggested a preferential utilization by the young adult of triglycerides composed of saturated fatty acids.

In vitro transformations of the insect plasmatocyte in some insects

In vitro transformation of insect plasmatocyte have been studied in physiological saline-versene, tissue culture, and the haemolymph. Transformation of a plasmatocyte into a granular cell can occur either by fragmentation of portions of the cytoplasm or by a gradual rounding-off of the fusiform plasmatocyte. The spherule cell is derived either from a plasmatocyte directly or from a granular cell. Cystocytes are formed from round plasmatocytes or from granular cells. Although gradual transformation of a plasmatocyte and a spherule cell into an adipohaemocyte was not observed, a granular cell was found changing into an adipohaemocyte. Oenocytoids are formed from fusiform plasmatocytes by cytoplasmic fragmentation. Podocytes and vermiform cells are derived from fusiform plasmatocytes by gradual transformation. All these transformations seem to be governed by specific physiological conditions. Oenocytoid- and lamellocyte-like cells are present in the haemolymph of Periplaneta americana (L.). The polymorphic plasmatocyte is the basic type of haemocyte in insects.

Modifications of the ionic content of the haemolymph and of the activity of Periplaneta americana in relation to diet

The concentration of K +, Na +, and Ca 2+ ions in the haemolymph of Periplaneta americana is compared for insects fed on different diets. The Na +/K + ratio is the same when the insect is fed on milk powder or wheat flour; it is lower on a diet of lettuce, the sodium ion concentration decreasing and potassium increasing. The activity of insects fed on milk powder is greater than that of the insects fed on lettuce. This result can be interpreted as being due to modifications in the potassium ion concentration of the haemolymph.