The control of Monomorium pharaonis (Hymenoptera: Formicidae) with Bacillus thuringiensis

In this study, the effect of different preparations made from Bacillus thuringiensis var. thuringiensis (strains: CCEB 555 and CCEB 058) on ants, Monomorium pharaonis, under laboratory conditions is reported. The different preparations tested consisted of (1) a liquid culture of the strain B. thuringiensis CCEB 555 (containing spores and exotoxin), (2) the supernatant of the culture broth of strain CCEB 555 (containing exotoxin), and (3) the biological preparation “Bathurin” prepared from the strain B. thuringiensis CCEB 058 (containing spores and inclusions, without exotoxin). The preparations were used either pure or in alternation with borax, i.e., 1 wk borax, 3 wk the respective preparation for several months. All preparations were found to be toxic to M. pharaonis and their effect was characterized by a slow extinction of the ant colony. Administration of “Bathurin” (1.3%) yielded a 100% mortality after 20 wk. Using a liquid culture of B. thuringiensis var. thuringiensis, 100% mortality was recorded after 21 wk, a period of time which did not differ from that obtained with the supernatant of the culture containing exotoxin. The alternation with borax was found to accelerate ant mortality by 9–10 wk after administration. In all experiments, the worker ants died first, the queen ants surviving them by 1–3 wk.In experiments employing worker ants only, a 100 and 98% mortality, respectively, occurred within 3 wk after administration of a liquid culture of B. thuringiensis and “Bathurin” supplemented with borax.     

Uptake of bacterial DNA by Chlamydomonas reinhardi

Escherichia coli [³H]DNA supplied to vegetative cultures of wild-type (mt+) and CW15 (mt+; mutant lacking the cell wall) Chlamydomonas reinhardi could bind to the cell wall of the wild-type and to the cell membrane of CW15 mutant cells. The extent of this binding decreased with time and was to a large degree (over 90%) DNA-ase-sensitive. Nevertheless, about 0.01% of the bacterial DNA remained irreversibly associated with the cells when they reached stationary phase. The irreversible binding of the donor bacterial DNA to Chlamydomonas cells could be increased by treatment of the cultures with polycations such as DEAE-dextran, poly-L-lysine and poly-L-ornithine. Although the CW15 cells rapidly degraded bacterial DNA in the culture medium wild-type cells showed only a small effect on the molecular weight of the donor DNA.The acid-insoluble radioactivity irreversibly bound to WT (+) cells consisted mainly of oligonucleotides with a small proportion present as less depolymerized donor DNA. No radioactivity, however, was found to be associated with the recipient high molecular weight Chlamydomonas DNA.No labeled donor DNA could be recognized in the cells given bacterial [³H]DNA in early stationary phase. Instead, radioactivity found in Chlamydomonas DNA corresponded to reutilization of [³H]thymine derivatives released as a result of [³H] DNA degradation. No evidence for the integration of detectable amounts of donor DNA sequences into the host cell DNA was obtained.     

Echinostoma revolutum: Labeling of miracidia with radioselenium in vivo and assay for host finding

Radioactivity was incorporated into Echinostoma revolutum worms and eggs when ⁷⁵Semethionine was administered intraperitoneally to mice infected with the fluke parasites. The levels of incorporation of radioactivity increased in proportion to the amounts of radioselenium used. During the period 3–10 days p.i. the maximum egg-bound radioactivity was, in general, achieved 3 days after the administration of the radioisotope, but substantial radiolabeling was obtained at all isotope levels until Day 10. The radioactivity of the miracidium constituted 29–34% of that of the egg. The radiolabeling procedure did not interfere with the biological characteristics (behavioral activity, infectivity) of the radiolabeled miracidia. Thus, the use of such labeled miracidia for host-finding studies seems acceptable. A radioisotope tracer system for assaying E. revolutum miracidial host finding was described. This system employs exposure of the first intermediate host snail, Biomphalaria alexandrina, to radiolabeled miracidia. A linear proportionality was found to exist between the number of penetrating miracidia and the amount of snail-bound radioactivity. Thus, snail-bound radioactivity retained after exposure to radiolabeled miracidia can be used to measure miracidial host-finding capacity under various experimental conditions.     

Biosynthesis of steroidal withanolides in Withania somnifera

Administration of 24-methylene-cholesterol-[28-³H] to Withania somnifera, yielded [³H] radioactivity in the isolated withaferin A and withanolide D, whereas administered 24-(R,S)-methyl-cholesterol-[28-³H] was not incorporated into these compounds. 24-Methylene-cholesterol is, therefore, proposed as a sterol precursor of the withanolides. A novel procedure is described for the isolation of withanolides from W. somnifera. This method in conjunction with an improved procedure for administration of labelled sterols and mevalonolactone produces a greatly increased yield of labelled withanolides.     

Metabolism of [6-14C]orotate by shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus

Incorporation of radioactivity from [6-¹⁴C]orotate into the pyrimidine constituents of shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus was examined with special reference to the unusual pyrimidine constituents. With each species, although 80% of the orotate supplied was catabolized to β-alanine, all the pyrimidine derivatives became radioactively labelled. With Pisum, the major part of the radioactivity incorporated into pyrimidines was located in UMP and the uracil derivatives, including the uracilyl amino acids willardiine and isowillardiine. With Phaseolus, UMP and the uracil derivatives were again the major radioactive products; incorporation of radioactivity into 5-ribosyluracil (pseudouridine), which accumulates in Phaseolus tissues, was comparable to the incorporation into orotidine and twice that found in cytidine. Lathyrus incorporated a substantially larger part of the presented [6-¹⁴C] orotate into pyrimidine derivatives than did the other two species. CMP was the most highly radioactive product, followed next by lathyrine and UMP. Surprisingly, 20% of the total radioactivity incorporated into pyrimidines by Lathyrus was located in the pyrimidine amino acid lathyrine. This confirms previous evidence that lathyrine is essentially a product of the orotate pathway. The overall recovery of radioactivity in all three species was 93–95%. The data emphasize the necessity of including the less common pyrimidine constituents, as well as the common ones, in quantitative studies of pyrimidine metabolism in plants.     

Humoral immune response of Galleria mellonella larvae after infection by Beauveria bassiana under optimal and heat-shock conditions

Natural infection of Galleria mellonella larvae with the entomopathogenic fungus Beauveria bassiana led to antifungal, but not antibacterial host response. This was manifested by induction of gallerimycin and galiomicin gene expression and, consequently, the appearance of antifungal activity in the hemolymph of the infected larvae. The activity of lysozyme increased at the beginning of infection and dropped while infection progressed. Exposure of the naturally infected animals to 43 °C for 15 min extended their life time.Galleria mellonella larvae were injected with 10⁴, 10⁵ and 10⁶ fungal blastospores, resulting in the appearance of strong antifungal activity and a significant increase in lysozyme activity in larval hemolymph after 24 h. Antibacterial activity was detectable only when 10⁵ and increased when 10⁶ blastospores were injected. The number of the injected B. bassiana blastospores also determined the survival rate of animals. We found that exposure of the larvae to 38 °C for 30 min before infection extended their life time when 10³ and 10⁴ spores were injected. The increase in the survival rate of the pre-heat-shocked animals may be explained by higher expression of antimicrobial peptides and higher antifungal and lysozyme activities in their hemolymph in comparison to non-heat-shocked animals.     

Biosynthesis of unusual acyclic isoprenoids in the Alga Botryococcus braunii

A ‘resting state’ isolate of the hydrocarbon-producing alga Botryococcus braunii photoassimilated sodium [¹⁴C]bicarbonate at rates comparable to fast growing algae, such as Chlorella (> 1.50 μg atoms ¹⁴C/mg chlorophyll·hr). Early in the reaction (up to several min), most of the radioactivity was associated with water-soluble metabolites. However, labelling of hexane-soluble compounds steadily from ca 3% at 15 sec to over 50% of the total incorporated ¹⁴C at 60 min. The purified hexane fraction, which consisted of a series of botryococcenes and squalene, constituted a relatively constant proportion (40–45%) of the total hexane-soluble radioactivity at all but the earliest time points (< 60 sec). This fraction initially consisted almost exclusively of a C₃₀ botryococcene (ca 91%) and squalene (ca 8%); however, small amounts of radioactivity sequentially appeared in the C₃₁, C₃₂ and C₃₄ botryococcenes. The results of pulse-chase experiments implicated the C₃₀ botryococcene as the precursor of the higher homologues; during the chase, loss of radioactivity from the C₃₀ compound was accompanied by a concomitant increase in the labelling of the C₃₁ and C₃₂ compounds. This study provides further evidence that the relatively slow growth of Botryococcus in culture may result, in part, from the diversion of a large proportion of reduced carbon into energetically expensive compounds and that the slower growth rate in the ‘resting state’ cannot be totally attributed to an impaired or intrinsically slow metabolism.     

A membrane component of the cellular slime mouldDictyostelium discoideum rapidly labelled with [32P]orthophosphate

After labellingDictyostelium discoideum (Strain Ax-2) for 30 min with [³²P]-orthophosphate a material was observed in cytoplasmic extracts, which cosedimented with polyribosomes in sucrose density gradients. The radioactivity in this material was insensitive to ribonuclease and deoxyribonuclease but was partially solubilized by alkali. All the radioactivity was rendered soluble in trichloroacetic acid by treatment with sodium deoxycholate. Most of the³²P counts were extractable in chloroform-methanol (2:1, v/v) and upon analysis by thin-layer chromatography the isotope was found in various phospholipids, chiefly phosphatidylethanolamine with some in lecithin and phosphatidylserine. Upon examination in an electron microscope the material was found to be composed of membrane, glycogen and an unidentified amorphous material.     

An evaluation of lipid metabolism in the insect trypanosomatid Herpetomonas muscarum uncovers a pathway for the uptake of extracellular insect lipoproteins

Lipid uptake and metabolism by trypanosomatid parasites from vertebrate host blood have been well established in the literature. However, there is a lack of knowledge regarding the same aspects concerning the parasites that cross the hemolymph of their invertebrate hosts. We have investigated the lipid composition and metabolism of the insect trypanosomatid Herpetomonas muscarum by ³H- palmitic acid and phosphate (³²Pi) and the parasite interaction with Lipophorin (Lp) the main lipid carrying protein of insect hemolymph. Gas chromatography-mass spectrometry (GC–MS) analyses were used to identify the fatty acids and sterols composition of H.muscarum. Furthermore, we investigated the Lp binding site in the plasma membrane of parasite by Immunolocalization. We showed that H. muscarum incorporated 3H-palmitic acid and inorganic phosphate (32Pi) which were readily used as precursor molecules of lipid biosynthetic pathways. Furthermore, H. muscarum was able to take up both protein and lipid moieties of Lp which could be used as nutrient sources. Moreover, we have also demonstrated for the first time the presence of a Lp binding site in the membrane of a parasite. Such results point out the role of describing the metabolic pathways of trypanosomatids in order to provide a better understanding of parasite-host interaction peculiarities. Such studies may enhance the potential form the identification of novel chemotherapeutic targets in harmful parasites.     

Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria

Heat-stable exotoxin production by 740 strains of Bacillus thuringiensis and related bacteria was investigated using the housefly, Musca domestica, from the following viewpoints: (1) the relation-ship between B. thuringiensis flagellar (H) serotypes and exotoxin production and (2) the exotoxin production by Bacillus species other than B. thuringiensis. Of 437 isolates belonging to 11 serotypes of B. thuringiensis which had been confirmed to produce parasporal inclusions, 35 isolates belonging to serotypes 1, 3a:3b, 4a:4c, and 10 produced heat-stable exotoxin. Exotoxin was not detected in the isolates of serotypes 3a, 4a:4b, 5a:5b, 5a:5c, 6, 7, and 8a:8b. No heat-stable exotoxin was demonstrated in 28 acrystalliferous isolates which possessed H antigens of B. thuringiensis serotypes 1, 3a, 4a:4b, 4a:4c, 5a:5c, 6, 7, 10, 11a:11c, and 12. A total of 270 B. cereus isolates which did not possess B. thuringiensis H antigen were examined and three isolates were found to produce heat-stable exotoxin. No heat-stable exotoxin was produced by B. subtilis (two strains), B. natto (one strain), and B. megaterium (two strains). These results indicate that the heat-stable exotoxin production in B. thuringiensis is a strain-specific property rather than a serotype(subspecies)-specific property.     

Metabolism of U14C leucine and U14C valine by adult female Glossina morsitans during pregnancy

After U¹⁴C leucine or U¹⁴C valine injections into haemolymph of adult female Glossina morsitans during late pregnancy, radioactivity was detected in the post-parturient female and its larval offspring in the injected material, lipids, and a range of non-essential amino acids. The level of radioactivity recorded from the third instar larva was higher than that remaining in the injected adult, and the activity was higher in amino acids than in the lipid fraction. Radiometric analysis of oöcyte and intra-uterine progeny 24 hr after haemocoelic administration to females of labelled leucine or valine revealed a pattern of radioactivity coincident with growth characteristics of these young stages. Rate of leucine uptake by the in utero third instar larva was slightly higher than that of valine, and this instar continues feeding even only a few hours before parturition. For both labelled materials, expired carbon dioxide and excreta from remales in early pregnancy showed significantly higher radioactivity than those in late pregnancy. Uric acid is the main nigrogenous waste of leucine and valine metabolism, though small amounts of these amino acids are also lost during excretion, with valine elimination being higher than leucine.     

In vitro and in vivo biosynthesis of xanthophylls by the cyanobacterium Aphanocapsa

Of the six carotenoids identified in the cyanobacterium Aphanocapsa, β-carotene, zeaxanthin, echinenone and myxoxanthophyll are the major pigments, whilst β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene are present only in trace amounts. With the exception of zeaxanthin, the other xanthophylls could be formed in vitro from [¹⁴C]phytoene in high yields, especially β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene. In a time course experiment of xanthopyll biosynthesis the flow of radioactivity from [¹⁴C]phytoene was followed through the pools of phytofluene, lycopene, and β-carotene. The reaction sequence from phytoene to xanthophylls is sensitive in vitro to both difunone, an inhibitor of carotene desaturation, and CPTA, an inhibitor of cyclization.     

Leukocytes of Biomphalaria glabrata: Morphology and behavior of granulocytic cells in vitro

Blood cells from Biomphalaria glabrata hemolymph were studied by phase microscopy in vitro. The most common cell is a granulocytic leukocyte, present in the hemolymph at a concentration of 334 ± 105 cells/mm³. This cell ranges in length from 14.1 ± 2.8 μm (including pseudopodia), when suspended in hemolymph, to 77.2 ± 15.1 μm, when allowed to spread on a glass substrate. Cytoplasmic inclusions and other organelles are described. Observations on the behavior of these granulocytic leukocytes in vitro confirm that a strong phagocytic response develops toward a variety of particles in the virtual absence of snail hemolymph.     

Two new subspecies of Bacillus thuringiensis isolated in Japan: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) and Bacillus thuringiensis subsp. tochigiensis (serotype 19)

Bacteriological and serological characteristics of three Bacillus thuringiensis isolates obtained in Japan were investigated. They formed typical rhomboidal parasporal inclusions but flagellar (H) antigens of these isolates were different from those of the known 17 H serotypes of B. thuringiensis. The three isolates were divided into two new serotypes (serotypes 18 and 19). The serotype 18 isolate (3–71) produced thermostable exotoxin and the inclusions of this isolate were toxic to larvae of the silkworm, Bombyx mori, but nontoxic to larvae of the mosquito, Aedes aegypti. The other isolate (119-72) belonging to serotype 18 produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. However, other bacteriological properties of the isolate 119-72 were similar to those of the isolate 3–71. The serotype 19 isolate (117-72) produced inclusions nontoxic to larvae of B. mori and A. aegypti and did not produce thermostable exotoxin. Acid production from saccharose and the production of brownish purple pigment were observed in the two serotype 18 isolates, while neither of them was observed in the serotype 19 isolate. In other 29 biochemical properties tested, there was no difference among the three isolates. Based on these characteristics, the following two subspecies names are proposed: Bacillus thuringiensis subsp. kumamotoensis (serotype 18) for the type strain 3–71 and Bacillus thuringiensis subsp. tochigiensis (serotype 19) for the type strain 117-72.     

Absorption and metabolism of dietary myo-inositol in Pieris brassicae

Absorption and metabolism of inositol was examined in Pieris brassicae fed myo-[2-³H] inositol or myo-[U-¹⁴C] inositol in the larval diet. Soluble and lipid-linked inositol were both labile in midgut tissue, but the soluble inositol pool appeared to be responsible for absorption across the midgut mucosa. The data suggest uptake of inositol from the mucosa into hemolymph and from hemolymph into tissues against a concentration gradient. Hemolymph radioactivity was mainly (98%) water-soluble. Phosphatidylinositol was observed in all tissues of P. brassicae examined, and was identified in the midgut mucosa of five other species of Lepidoptera. In most tissues, with the exception of larval midgut and adult abdomen, lipid-linked inositol accounted for a minor proportion of total tissue inositol, e.g. in the testis and ovaries of 2–4-day-old adults c. 75% of radioactivity was recovered from the soluble inositol pool. There was a marked loss of total body radioactivity during metamorphosis: 3-day-old post emergence adults retained only about 30% of the radioactivity present in perpupae. The titer of the soluble inositol pool was maintained relatively unchanged throughout the pupal stage, but lipid-linked radioactivity declined: 10-day-old pupae contained less than 50% of the lipid-linked inositol radioactivity observed in prepupae.     

Influence de la température sur la biosynthèse des acides gras de deux champignons filamenteux, Aspergillus ochraceus et Mucor mucedo

The two moulds, Mucor mucedo and Aspergillus ochraceus, grow very rapidly at 22°; when transferred to 12°, Mucor shows a regular growth and sporulation while Aspergillus development is almost entirely inhibited. ¹⁴C Acetate was supplied to both fungi at 12° and 22°. In both, the total radioactivity of saturated fatty acids was less important at the lowest temperature. In contrast, C 18:1 radioactivity rose in Mucor while it was C 18:2 radioactivity which rose in Aspergillus at the lowest temperature. There was an increase in phosphatidylethanolamine (PE) and phosphatidylcholine (PC) radioactivities in Mucor while the phosphatidylserine (PS) radioactivity decreased at low temperature. By contrast, when Aspergillus was placed in the same cultural conditions, PE and PC radioactivities decreased while PS radioactivity increased. Based on these results, a tentative explanation of the improved cold-resistance of M. mucedo is presented.     

The granular cells of Galleria mellonella during clotting and phagocytic reactions in vitro

Light and electron-microscopic observations of the blood-cells (hemocytes) of the wax-moth Galleria mellonella showed that hemolymph coagulation was initiated by the rapid release of material from the granular cells. During incubation for short terms in vitro these cells showed progressive degranulation as material derived from the granules was discharged into the hemolymph. Attempts to determine the nature of this material by staining with ruthenium red proved mainly unsuccessful. When challenged with bacteria in vitro the granular cells failed to phagocytose these particles and instead the bacteria became embedded in the granular material surrounding these cells. The mode of coagulation reported here is compared with previous reports of the role of invertebrate hemocytes in hemolymph clotting.     

Distribution of 14C-l-leucine in organs and tissues of guinea pigs infected with tRichostrongylus colubriformis

Earlier experiments with intestinal nematode infection which had shown changes to skeletal muscle and liver protein metabolism, did not examine the metabolism of the gastrointestinal tract nor attempt to integrate these changes with the whole body. Consequently the distribution of ¹⁴C-l-leucine in all organs and tissues of guinea pigs infected with Trichostrongylus colubriformis was compared with uninfected animals fed either ad libitum or quantitatively reduced rations.There were no differences between experimental groups in total radioactivities recovered, but in the infected animals radioactivity accumulated in the liver, stomach and small intestine, and caecum and large intestine at the expense of the eviscerated carcase and skin. Reducing the ration of uninfected guinea pigs did not affect the distribution of leucine, apart from reducing the fraction in the eviscerated carcase. Incorporation of ¹⁴C-l-leucine and its relationship to protein synthesis in the livers and eviscerated carcases of the three experimental groups is discussed. It was concluded that events in the small and large intestines, which are independent of anorexia, are important components of protein metabolism in intestinal nematode infection.     

The delta-endotoxin of Bacillus thuringiensis. V. On the occurrence of endotoxin fragments in hemolymph

Leucine-³H labeled crystals of Bacillus thuringiensis δ-endotoxin were fed to last-instar larvae of spruce budworm, Choristoneura fumiferana, eastern forest tent caterpillar, Malacosoma disstria, and silkworm, Bombyx mori. Radioactivity was detected in hemolymph 1 min after feeding in the first two species, but not until 3–5 min after feeding in silkworm larvae. Most of the radioactivity from hemolymph of all three species eluted from gel filtration columns at the same elution volume indicating similar moleculear weights (<1800 daltons).     

Persistence of Bacillus thuringiensis in foundation beeswax and beecomb in beehives for the control of Galleria mellonella

Beecomb was protected against wax moth attack by impregnating foundation beeswax with Bacillus thuringiensis. Bakthane, a serotype I product, gave good protection of broodcomb for 2 yr at 0.5% in wax and partial protection for 6 yr at 2%. The active component was exotoxin present at about 0.9% in Bakthane in a very insoluble state. No harm to the bees was detected, even when a partially purified preparation of exotoxin was used, but further tests on the leaching of pure exotoxin from comb into honey are required before the latter can be regarded as a practical method of wax moth control. The action of exotoxin on Galleria mellonella larvae was slower than that of the spore-crystal complex and less than that of exotoxin on some Diptera. Spores and crystals of serotype V were × 500 as potent in G. mellonella as those of serotype I and exotoxin on honey-rich artificial food in laboratory assays, but their activity deteriorated in the first hive trials with treated foundation wax. Prehive deterioration was due both to Teepol and soap in the liquid lubricating the wax mill rollers and to moist storage of the foundation. This deterioration was prevented by the use of Triton X-100 as lubricant, by drying the newly impregnated foundation wax and by storing it in dry conditions. This resulted in good protection of comb for one hive season by 1% of serotype V Thuricide in foundation wax. However, protection after two seasons varied, making the use of serotype V spores and crystals uneconomical for commercial practice, although safe for bee and man.