Host regulation and the embryonic development of the endoparasitoid Toxoneuron nigriceps (Hymenoptera: Braconidae)

Insect endoparasitoids modulate the host physiology through the injection of maternal-derived substances into the host, inducing physiological and hormonal changes in the host's internal environment to benefit parasitoid development. These changes are direct to control host development and regulate nutrient availability to the developing parasitoid, and they are synchronized with parasitoid development. Eggs of some of these parasitoids have low yolk content and require nutrients from the host hemolymph to initiate and complete embryogenesis. We report changes in the amino acid composition and protein profile of the host hemolymph of the endoparasitoid Toxoneuron nigriceps, and improved the in vitro culture of pre-germ band stage eggs. The protein profile of parasitized larvae was similar to controls throughout the embryonic development, but total amino acid concentration decreased in the first 2 h after parasitization, significantly increasing in the following hours up to 8 h. Amino acid levels were higher in parasitized larvae from 16 to 28 h after parasitization. Comparison of single amino acids indicated amino acids involved in energy metabolism (Krebs cycle) followed a trend during parasitoid embryogenesis, and their changes were correlated with embryonic development. Improvement in the in vitro development of 6 h-old eggs of T. nigriceps was obtained by adding factors released by the host fat body to the artificial medium, while a cell lysate stimulated embryogenesis and allowed the full development of newly laid eggs in vitro.     

Major hemolymph proteins from larvae of the black swallowtail butterfly,Papilio polyxenes

Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin-I and apolipophorin-II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin-I and apolipophorin-II, respectively, revealed cross-reactivity of apoLp-I with Manduca sexta apoLp-I, and apoLp-II with M. sexta apoLp-II. Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein-1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein-2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.     

Identification of an Arylphorin-type Storage Protein in the Sweet Potato Hornworm, Agrius convolvuli

A major hemolymph protein (M_r 480,000) in the larvae of the sweet potato hornworm, Agrius convolvuli, was purified and characterized. This protein was isolated with a high yield from the hemolymph of day 3 fifth final instar larvae by ammonium sulfate precipitation and Phenyl-Sepharose and Q-Sepharose column chromatographies. The protein has two subunits, an M_r 84,000 subunit (α) and an M_r 80,000 subunit (β), and the native protein was composed of a heterohexamer (α₃β₃). The two subunits have similar amino acid compositions, with high contents of aromatic amino acids (about 15% phenylalanine plus tyrosine) and low levels of methionine. The N-terminal amino acid sequences of both subunits showed high homologies with insect arylphorin-type storage proteins. The protein concentration in the hemolymph increased steeply from day 3 final instar larva and reached a maximum level of 42 mg/ml in females and 41 mg/ml in males among wandering larvae. The concentration in the hemolymph declined once during the larval–pupal transformation but remained high during the early–mid pupal period and almost disappeared after adult emergence. These quantitative changes were the same for males and females. Based on these characteristics, we identified the hemolymph protein as an arylphorin-type storage protein.     

Changes of the Antioxidant Status and System of Generation of Free Radicals in Hemolymph of Galleria mellonella Larvae at Microsporidiosis

Changes of superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities as well as of the content of SH-containing compounds were revealed in hemolymph of the native and the Vairimorpha ephestiae microsporidian-infected greater wax moth Galleria mellonella larvae. The SOD and GST activities in hemolymph of infected insects decreased at the stage of merogony, whereas during massive sporulation the enzymatic antioxidant activity in host tissues was higher than in control. By the ESR spectroscopy method, using the 1-hydroxy-3-carboxy-pyrrolidinespin-trap, generation of free radicals in hemolymph of infected insects was shown to decrease only at the stage of sporogony. The phenoloxidase activity in lymph was lower at acute microsporidiosis than in native larvae. The hemolymph concentration of thiol-containing proteins in infected insects did not differ from that in control. We suggest that decrease of generation of free radicals in hemolymph of the greater wax moth larvae at the stage of sporogony is due to a suppression of the prophenoloxidase system and an elevation of the antioxidant activity.     

Purification and Properties of Ferritin from the Last Instar Larval Hemolymph of Protaetia brevitarsis (Coleoptera)

Ferritin, an iron storage protein, has been purified from the last larval hemolymph of Protaetia brevitarsis (coleoptera) by KBr density gradient ultracentrifugation and resource Q (anion exchange chromatography) using fast performance liquid chromatography (FPLC) system. The iron content of ferritin was determined by atomic emission spectroscopy and FereneS stain. Ferritin of P. brevitarsis is shown to have two different subunits presented on a SDSPAGE in normal (N) and ironinjected (I) hemolymph. SDS PAGE revealed that the ferritin consists of two major polypeptides of 27 and 28 kDa in normal hemolymph. Interestingly, however, 30 kDa subunit was substituted for 28 kDa when iron was injected into the hemolymph. Apporximate isoelectric points of 27 kDa, 28 kDa, and 30 kDa ferritin subunits were 6.7, 6.75, and 6.8, respectively. Ferritin of P. brevitarsis was detected by FereneS stain and confirmed by Western blotting using its polyclonal antibody. Other characteristics such as amino acid composition and Nterminal amino acid sequence were investigated. Amino acid composition of ferritin (N and I) was rich in alanine, glycine, glutamine or glutamic acid and serine, but poor in histidine, arginine, methionine and phenylalanine.     

Changes in the lipid and fatty acid composition of hemolymph and ovaries during the reproductive cycle of Labidura riparia

Lipid metabolism was investigated during the reproductive cycle of Labidura riparia (Pallas). The lipid classes and their constitutive fatty acids present in hemolymph and ovaries were measured using thin‐layer chromatography and gas‐liquid chromatography. In the hemolymph, total lipids increase steadily from the previtellogenic period to vitellogenic arrest. These lipids are predominantly diacylglycerols and phospholipids. In the ovaries, total lipids increase during vitellogenesis then decrease during the vitellogenesis arrest period. The major lipids are triacylglycerols, followed by phospholipids. In both hemolymph and ovaries, all lipid classes contained variable proportions of seven main fatty acids: the saturated fatty acids myristic acid (14:0), palmetic acid (16:0), and stearic acid (18:0); the monounsaturated fatty acids palmitoleic acid (16:1) and oleic acid (18:1); and the polyunsaturated fatty acids linoleic acid (18:2) and linolenic acid (18:3). Unsaturated fatty acids predominate throughout the reproductive cycle. The percentage compositions of total and triacylglycerol fatty acids do not change markedly during the reproductive cycle in hemolymph nor in ovaries, with 18:2, 18:1 and 16:0 fatty acids being the major components. However, for diacylglycerols and phospholipids, the proportions of fatty acids vary systematically. For phospholipids during the vitellogenesis period, 18:2 increases considerably whereas other fatty acids decrease; for diacylglycerols, these fatty acids vary in the reverse way.     

A high molecular weight diapause-associated protein from the stem-borer Busseola fusca: Purification and properties

The hemolymph of diapausing larvae of the stem borer, Busseola fusca Fuller (Lepidoptera: Noctuidae), contains an electrophoretically distinct protein band on nondenaturing polyacrylamide gels. The protein, called the Busseola diapause protein (BDP), was purified by a combination of density gradient ultracentrifugation, gel permeation, and affinity chromatography. It is a high molecular weight protein (M_r ～5 × 10⁵; pl = 6.1) that is composed of two subunits, I (M_r ～88,000 ± 4,000) and II (M_r ～79,000 ± 1,000), which are not linked by disulfide bridges. The protein contains both lipids (2%) as well as covalently bound carbohydrates (1%). The inability to stain the fluorescein isothiocyanate-conjugated concanavalin A (FITC-Con A) suggests that the carbohydrate moiety of BDP is not of the high mannose type. Amino acid analysis showed a high tyrosine plus phenylalanine content (16 mol%). Labeling studies using [³⁵S]-methionine showed that de novo synthesis by the fat body tissue occurs only in diapausing larval insects. It is proposed that the BDP could serve a storage function by providing the amino acids needed for the synthesis of pupal and adult structures.     

The effect of nucleopolyhedrovirus infection and/or parasitism by Microplitis pallidipes on hemolymph proteins, sugars, and lipids in Spodoptera exigua larvae

We determined the physiological effects of joint and separate nucleopolyhedrovirus (NPV) infection and parasitism by the endoparasitoid Microplitis pallidipes Szepligeti on biochemical events in the noctuid Spodoptera exigua (Hübner). The results indicated that in parasitized larvae, compared to healthy larvae, total protein concentration in host hemolymph began to decline and total sugar concentration significantly increased by the first day, while lipid content in the host body significantly increased by the second day after parasitization. Meanwhile, in jointly infected and parasitized hosts, compared to parasitized larvae, total protein concentration was consistently higher, total sugar concentration was consistently lower, and lipid content became higher by the second day after treatment. In virus-infected larvae, compared to healthy larvae, total protein concentration sharply declined during the first two days but increased by the third, while total sugar concentration increased on the second and third days after virus infection but decreased at other observation times, and lipid content began to increase by the second day after virus infection. Finally, in larvae that were both parasitized and virus-infected, compared to just virus-infected larvae, total protein concentration increased during the first two days but decreased by the third, total sugar concentration increased only on the first and fourth days, and lipid content decreased significantly on the first day but began to increase by the second day after treatment. These findings led us to conclude that parasitization inhibited protein mobilization but stimulated sugar mobilization in host hemolymph, and promoted lipid mobilization in the host body, while Spodoptera exigua NPV infection stimulated protein mobilization induced by parasitization but inhibited sugar mobilization induced by parasitization.     

The reproduction strategy of oyster ostrea edulis L. from the biochemical point of view

• 1.1. Influence of the biochemical composition of food (four species of micro-algae and one mixture) on the biochemical composition of gonads and larvae of O. edulis (total protein, lipid, carbohydrate, ash content, neutral and polar lipid class composition, amino acid composition and fatty acid composition of total, neutral and polar lipids) and the size of newly released larvae have been investigated.
• 2.2. Precentage of total lipids and triacylglycerols in gonads depends on that in algae (r = 0.52 and 0.69 accordingly).
• 3.3. Gonads rich in lipids had a higher level of triacylglycerols, phospholipids, polar lipids and a lower value of the ratio phosphatidylethanolamine/phosphatidylcholine (PE/PC) than gonads with a low lipid content.
• 4.4. Amino acid composition of gonads depends on that of food, in this case, essential acids are preferentially accumulated (Asp acid, Ser, Ala, Cys, Tyr and Pro) and two non-essential (Thr and Lys).
• 5.5. Fatty acid composition of total lipids of gonads was rather stable; except for the two essential acids 20:523 and 22:6w3, their percentage depends on that of food r = 0.65 and 0.65 accordingly). Fatty acid composition of neutral lipids was more diverse (in number and degree of variety) as compared to polar lipids.
• 6.6. Larvae released from oysters with gonads rich in lipids had a higher percentage of lipids, triacylglycerols, size and a lower ash percentage and value of ratio PE/PC, as compared to larvae from gonads with low lipid content. Total lipid and triacylglycerol contents in gonads correlate rather well with those in larvae (r = 0.77 and 0.47 accordingly).
• 7.7. Phospholipid class composition of larvae strongly depends on that of gonads. All the correlations are high and positive in character (except for phosphatidylinositol).
• 8.8. Amino acid composition of larvae depends on that of gonads and, as in the case with gonads, the same essential acids are accumulated in the first place.
• 9.9. Fatty acid composition of total lipids of newly released larvae was rather stable and independent on that of gonads except for total polyunsaturated acids (r = 0.70) and 20:5w3 (r = 0.65). Fatty acid composition of neutral lipids was lesser diverse (in number and degree of variation) as compared to polar lipids.

     

Isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of Manduca sexta larvae

A protein that inhibits hemocyte aggregation has been isolated from hemolymph of Manduca sexta larvae and named hemocyte aggregation inhibitor protein (HAIP). HAIP has a M_r = 50,000, pI = 8.5, and contains 7% carbohydrate. It is present at 230 ± 20 μg/ml in hemolymph of day 3 fifth instar larvae. Antibodies to HAIP do not cross-react with M. sexta hemolin, which is similar in size and charge and also inhibits hemocyte aggregation. HAIP and hemolin have some similarity in amino acid composition and NH₂-terminal sequence, but are different in overall secondary structure, as determined by CD spectroscopy. The concentration of HAIP in hemolymph is not affected by injection of larvae with bacteria. A protein of approximately 50,000 daltons that reacts with antibody to M. sexta HAIP is present in hemolymph of Bombyx mori, Heliothis zea, and Galleria mellonella. Although the function of HAIP in vivo is not yet clear, it may have a role in modulating adhesion of hemocytes during defensive responses. © 1994 Wiley-Liss, Inc.     

CHANGES IN THE HEMOLYMPH PROTEIN PROFILES IN Galleria mellonella INFECTED WITH Bacillus thuringiensis INVOLVE APOLIPOPHORIN III. THE EFFECT OF HEAT SHOCK

This report concerns the effect of heat shock on host–pathogen interaction in Galleria mellonella infected with Bacillus thuringiensis. We show enhanced activity against Gram‐positive bacteria in the hemolymph of larvae pre‐exposed to heat shock before infection with B. thuringiensis. Heat shock influenced the protein pattern in the hemolymph of infected larvae: more peptides with a molecular weight below 10 kDa were detected in comparison with nonshocked animals. Additionally, we noticed that the amount of apolipophorin III (apoLp‐III) in the hemolymph decreased transiently following infection, which was considerably higher in larvae pre‐exposed to heat shock. On the other hand, its expression in the fat body showed a consequent infection‐induced decline, observed equally in shocked and nonshocked animals. This suggests that the amount of apoLp‐III in the hemolymph of G. mellonella larvae is regulated at multiple levels. We also report that this protein is more resistant to degradation in the hemolymph of larvae pre‐exposed to heat shock in comparison to nonshocked larvae. Two‐dimensional analysis revealed the presence of three isoforms of apoLp‐III, all susceptible to proteolytic degradation. However, one of them was the most abundant, both in the protease‐treated and untreated hemolymph. Taking into consideration that, in general, apoLp‐III has a stimulative effect on different immune‐related hemolymph proteins and peptides, the reported findings bring us closer to understanding the effect of heat shock on the resistance of G. mellonella to infection.     

The effect of L-canavanine and L-canaline on the hemolymph amino acid composition of the tobacco hornworm,Manduca sexta (L.) (Sphingidae)

Tobacco hornworm larvae, Manduca sexta (L.) (Sphingidae), were administered L-canaline either by parenteral injection or by dietary consumption. The overt toxicity and the alteration of hemolymph amino acids caused by these nonprotein amino acids were evaluated. The LD₅₀ value for parenterally administered canavanine and canaline is 1.0 and 2.5 mg/g fresh body weight, respectively. A dietary concentration of 5.2 mM for canavanine and over 20 mM for canaline represent the respective LC₅₀ values. A large percentage of the larvae reared on diets supplemented with additional arginine, ornithine, or 2,4-diaminobutyric acid in addition to canavanine or canaline were unable to complete larval-pupal ecdysis. These toxic effects were associated with a decreased glutamic acid hemolymph titer and dramatically elevated ornithine. On the other hand, larvae administered canavanine or canaline alone, either by dietary consumption or parenteral injection, experienced less drastic developmental aberrations. These symptoms were in some cases correlated with increased ornithine and glutamic acid titers. Evidence is presented that even a canavanine- and canaline-sensitive insect such as M. sexta has a marked ability to eliminate these protective allelochemicals.     

Alteration of hemolymph polypeptides in Manduca sexta larvae parasitized by Cotesia congregata: A two-dimensional electrophoretic analysis and comparison with major bacteria-induced proteins

Analyses using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) previously demonstrated that parasitization by the braconid wasp Cotesia congregata significantly alters the normal hemolymph polypeptide profile of host Manduca sexta larvae. In the present study two-dimensional gel analyses corroborated our earlier findings and provided additional evidence that multiple parasitism-specific polypeptides were induced, which varied according to the stage of development of the wasps. Parasitization additionally elicited changes in the total protein concentration detected in the blood. Initially an elevation was observed, with newly parasitized larvae exhibiting a twofold elevation in hemolymph protein concentration by 12–24 h postoviposition. In contrast, terminal-stage hosts with second instar parasites had significantly less protein in the hemolymph, likely due to reduced growth and inhibition of arylphorin synthesis by the fat body during the final stages of parasitism. Comparison of the array of hemolymph polypeptides produced in unparasitized larvae injected with 10⁶cells of the gram-negative bacterium Enterobacter cloacae with those proteins induced by parasitization indicated the two classes are different. Our findings confirm that the hostresponse to parasitism is a specific one, and not mimicked by bacterial challenge. Duringshort-term in vitro culture of wasp larvae dissected from the host hemocoel, several proteins were detected in the medium using SDS - PAGE, with their appearance in vitro suggestive of secretion by the wasps in vivo. Moreover, hemolymph from the parasites had significant amounts of putative host proteins, including an arylphorin - like polypeptide and a protein with a mobility similar to that of insecticyanin. Thus, a dynamic interchange of proteins may occur, with the parasites accumulating host proteins while simultaneously secreting a variety of factors into the host hemocoel.     

Hemolymph amino acid variations following behavioral and genetic changes in individual Drosophila larvae

This study investigated the effect of different sampling environments on hemolymph amino acid content of individual Drosophila melanogaster larvae. Hemolymph was collected from individual third instar larvae under cold-anesthetized, awake, and stress conditions. Qualitative and quantitative hemolymph amino acid analyses were performed via capillary electrophoresis with laser-induced fluorescence detection. The hemolymph amino acid concentrations, particularly arginine, glutamate, and taurine, changed significantly depending on the prior-to-sample-collection environments. Hemolymph amino acid analyses of six different Drosophila genotypes including two control genotypes and four mutant alleles were also carried out. Two mutant genotypes with over and under expression of a putative cystine-glutamate exchanger subunit were significantly different from each other with respect to their hemolymph glutamate, glycine, lysine, and taurine levels. Hemolymph amino acid analyses of stressed larvae of two control and two mutant genotypes indicated that behavior-related hemolymph chemical changes are also genotype dependent.     

Properties of serum high-density lipoproteins in the crab, Cancer antennarius Stimpson

Two classes of high-density lipoprotein (HDL) comprise virtually all the lipoprotein mass in female hemolymph. These lipoproteins have hydrated densities of 1.187 g/ml (HDL3) and 1.112 g/ml (HDL2). A third species (HDL1, density 1.080 g/ml) appeared in ovigerous crabs. The mean annual HDL protein concentration was 109 mg/dl of which 67% was HDL3. HDL proteins of both HDL2 and HDL3 were mostly insoluble in tetramethylurea. Three major components with apparent mol. wts of 185,000, 100,000 and 84,000 daltons were identified by gel electrophoresis in SDS. Amino acid compositions are reported. Electron microscopy indicated that the HDL are polymorphic and discoidal. Similarities in shape and differences in size of HDL3 and HDL2 particles were consistent with their lipid and protein composition. Phospholipids, mostly phosphatidylcholine, were the dominant lipid class (74%); no cholesteryl esters were detected. Palmitic and oleic acids were the major fatty acid components of esterified lipids.     

Phospholipids from the hepatopancreas of Indian horseshoe crab Carcinoscorpius rotundicauda

Total phospholipids were extracted from the heart, hepatopancreas, and hemolymph of the Indian horseshoe crab Carcinoscorpius rotundicauda by the conventional method. Characteristic group reaction and 2-dimensional thin-layer chromatography on silica gel were used for identification of different phospholipids. The phospholipid profile obtained from hemolymph and 2 major organs are comparable and show phosphatidyl choline (PC) and phosphatidyl ethanolamine to be the major phospholipids. A phospholipid has been consistently detected migrating immediately below the PC in the thin-layer chromatogram of lipids extracted from the hepatopancreas. When mixed methyl esters of this slower moving PC are resolved on a silica gel plate ran in hexane ether:acetic acid 80:20:1, with appropriate controls, an additional spot is seen just below the normal methyl ester, indicating a difference between the fatty acid compositions of 2 PC (e.g., regular and slower). The slower mixed methyl esters were found to comprise mainly the 4 saturated fatty acids: lauric, myristic, palmitic, and stearic. The slow moving PC seems to consist mainly of molecular species with the above-mentioned saturated fatty acids at both Sn 1 and Sn 2 positions.     

Lack of interaction between the milky disease bacterium Paenibacillus popilliae and stilbene-derived optical brighteners in Japanese beetle larvae

Optical brighteners cansynergistically enhance nucleopolyhedrovirusinfectivity to lepidopteran larvae by blockingthe sloughing of infected primary midgut cellsand inhibiting the formation of the peritrophicmembrane in the hosts. Because of similaritiesin the route of infection, we investigatedwhether optical brighteners would also enhanceinfection with the milky disease bacterium,Paenibacillus popilliae, of Japanesebeetle, Popillia japonica, larvae. Thelarvae were kept in soil mixed with P.popilliae spore preparations and the opticalbrighteners Blankophor BBH, P167, or RKH withperennial ryegrass provided as food. Noenhancing effect of any of the opticalbrighteners on P. popilliae infection wasobserved at a concentration of 0.1% (w/w). Rather, when mixed into the soil at 0.02, 0.1,or 0.5% (w/w) BBH reduced P. popilliaeinfection at the highest rate.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

The effect of low oxygen concentration on the acyl-lipid and fatty-acid composition of the C4 plant Amaranthus paniculatus L.

Acyl lipids and their constituent fatty acids were studied in leaves, chloroplasts and bundle-sheath strands of the C₄ plant Amaranthus paniculatus L. grown under normal and 4%-oxygen-containing atmospheres. In all fractions the major lipids were found to be monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovo-syldiacylglycerol and phosphatidylglycerol. Significant quantities of phosphatidylcholine and phosphatidylethanolamine were restricted to leaves and bundle-sheath strands. All lipids, except phosphatidylglycerol where 3-trans-hexadecenoic acid was also present, contained palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. On a chlorophyll basis and compared with whole leaves, the amounts of phosphatidylcholine and phosphatidylethanolamine in bundle-sheath strands were considerably reduced. Three weeks after the change from a normal to a 4% atmospheric O₂ level, the galactolipid content, particularly in the bundlesheath strands, was enhanced. There were no significant differences in the degrees of saturationunsaturation of total acyl lipid for the plants grown in the low oxygen and normal atmospheres, although under 4% O₂ the phosphatidylglycerol contained an increased proportion of 3-trans-hexadecenoic acid at the expense of palmitic acid.Abbreviations DGDG digalactosyldiacylglycerol - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - SQDG sulphquinovosyldiacylglycerol     

Germination and outgrowth of Bacillus popilliae in hemolymph slide mounts

Cabbage looper hemolymph induced rapid germination and outgrowth of spores of Bacillus popilliae. Spores germinated within sporangia but outgrowth occurred from free released spores as well as from spores retained in sporangia. With 37°C, an alkaline pH, and tyrosinase, outgrowth resulted in 1 hr. Of six strains of milky disease bacteria tested, hemolymph mediated germination and outgrowth of only those which are infective perorally for European chafer larvae, indicating a potential use as a screening tool to assess virulence for the chafer.