Alanine aminotransferase in the three last larval instars of Barathra brassicae infected by Nosema plodiae

During three last larval instars of Barathra brassicae the alanine aminotransferase activity pattern exhibits two maxima which do not correspond with the maxima of activity of phosphatases and proteases. In the larvae infected by Nosema plodiae a different course of activity can be seen from the 5th day of infection. The activity pattern in the diseased larvae rises above that of a normal larva by 90% shortly before the last molt and reaches a maximum at the end of normal larval development. Alanine aminotransferase in the gut and in the fat body exhibits two main pH optima of activity, 8.0 and 9.5, respectively, and a low activity peak at pH 4.5. This last activity is not present in diseased animals. The temperature and pH inactivation are also described.     

Factors controlling in vitro hatching of Vairimorpha plodiae (Microspora) spores and their infectivity to Plodia interpunctella,Heliothis virescens, and Pieris brassicae

Factors which influence the hatching of spores and proliferation of stages of the microsporidium Vairimorpha plodiae in two susceptible insects, Plodia interpunctella and Heliothis virescens, and one nonsusceptible insect, Pieris brassicae, were investigated. Spores hatched in 0.1 and 1 m KCl solutions when subjected to a change in pH, from pH 11 to pH 8. K⁺ was essential for hatching; NaCl solutions were not effective. Ca²⁺ and Mg²⁺ inhibited hatching, and calcium and magnesium chelating agents enhanced it. All three insect species had alkaline midgut contents and smooth, fragile peritrophic membranes. Spores hatched inside the midguts of all three insect species (P. interpunctella: maximum rate, 92.5%; H. virescens, 91.5%; P. brassicae, 82%). Sporoplasms were observed in the midgut epithelial and associated tracheole cells of P. brassicae. Both H. virescens and P. brassicae became infected when injected intrahemocoelically with spores.     

Carbohydrate energy metabolism in Fasciola gigantica (trematoda)

Umezurike G. M. and Anya A. O. 1980. Carbohydrate energy metabolism in Fasciola gigantica (Trematoda). International Journal for Parasitology10: 175–180. Adult Fasciola gigantica contained 4.49 ± 0.06 % (mean ± S.D.) wet weight glycogen. Tissue homogenates contained high levels of malate dehydrogenase (MDH), NAD-linked malic enzyme (ME), Phosphoenolpyruvate carboxykinase (PEPCK) and lactate dehydrogenase (LDH). MDH, PEPCK and ME activities appeared to be localized in both cytosolic and mitoehondrial fractions, fumarase activity appeared to be predominantly mitochondrial whereas LDH and pyruvate kinase activities were cytosolic in distribution. Polyacrylamide gel electrophoresis revealed the predominance of LDH-1, LDH-2 and LDH-3 but only traces of LDH-4 and LDH-5 isoenzymes in the crude cytosolic fraction. LDH activity in the crude sample was inhibited by excess substrate (pyruvate). The mitoehondrial system showed NADH -cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase, NADH oxidase and some cytochrome c-oxygen oxidoreductase activities. Under anaerobic conditions, NADH-fumarate oxidoreductase and succinate-NAD + oxidoreductase activities of mitoehondrial preparations were stimulated in the presence of ADP and ATP respectively. Isolated mitochondria contained rhodoquinone and no ubiquinone, and isolated rhodoquinone was readily reduced by succinate in the presence of submitochondrial particles. Hydrogen peroxide was produced by submitochondrial particles in the presence or absence of KCN or in the presence of fumarate.     

Trypanosoma cruzi: Lactate dehydrogenase isoenzymes and infections in mice

Total plasma LDH isoenzyme (EC 1.1.1.27) levels increased significantly over the normal level in mice infected with strains of Trypanosoma cruzi from three different geographic locations, but some strain differences were observed. The most rapid increase was exhibited by the blood-induced Tulahuen strain, but this strain, unlike the House 510 or House 11, did not elicit an increase during the early period of infection. Overall increases in LDH-1 and LDH-2, heart isoenzymes, were most marked in vector-derived House 510 infections, but, as in the Tulahuen strain, a considerable increase was also observed in blood-induced infections. The House 510 strain also elicited significant increases in LDH-4; these were particularly high during the early period of the blood-induced infection. By contrast, the vector-derived Tulahuen strain elicited a higher increase in LDH-4 during the early period than the House 510 or House 11 strains. Comparable similarites and differences were also observed in regard to LDH-3, 5, and “X.” The most marked isoenzyme increases were those of “LDH-X” exhibited by the blood-induced House 510 and vector-derived Tulahuen strains.Parallel histopathologic studies of liver, heart, and skeletal muscle disclosed significant pathology in all the infections. Animals with blood-induced Tulahuen strain infections characteristically showed extensive necrosis with marked multiplication of parasites throughout the liver, but little or no evident damage to the heart and skeleal muscle. Animals infected with House 510 and House 11 strains exhibited minimal pathology in the liver but severe damage to the heart and skeletal muscle. Increases in LDH-4 and LDH-5, isoenzymes which represent both liver and skeletal muscle, in blood-induced Tulahuen infections were attributed largely to liver damage, but in the House 510 and House 11 infections were related more to skeletal muscle.     

Transovarian transmission of Nosema plodiae in the Indian-meal moth, Plodia interpunctella

Twenty-five adult female Plodia interpunctella infected with Nosema plodiae laid 856 eggs in laboratory tests; 12.5% of the eggs were infected transovarially. The highest level of transmission by an individual female was 14 infected eggs of 27 laid (51.8%); the lowest level of transmission observed was 1 of 43 eggs (2.3%). All stages of N. plodiae were not transmitted with equal frequency; moreover, most eggs harbored predominently only the trophozoite stage of the pathogen. Approximately 80% of the infected eggs contained trophozoites almost exclusively; about 13.1% contained about an equal number of spores and trophozoites, and about 6.5% contained mainly spores. Histological observations indicated that infections may be initiated in nurse cells and subsequently transferred to associated oocytes.     

Different course of proteolytic inhibitory activity and proteolytic activity in Galleria mellonella larvae infected by Nosema algerae and Vairimorpha heterosporum

The activity of protease inhibitors and proteases was studied in the hemolymph, gut, and fat body of 7th-instar larvae of Galleria mellonella infected by two microsporidia, Nosema algerae and Vairimorpha heterosporum. The increase in inhibitory activity in the hemolymph was substantial, and coincided with the development of the disease. The increase in inhibitory activity in the gut was almost doubled by N. algerae as compared with V. heterosporum, whereas the increase in inhibitory activity in fat body was found only in V. heterosporum-infected larvae. The course of proteolytic activity followed an inverse pattern to the elevated activity of inhibitors in the gut and the fat body, and rose only in moribund larvae at the end of the course of V. heterosporum infection. The differences in the pattern of proteases and inhibitors reflect the organ specificity of each of the microsporidia.     

Absorption and transport of dietary lipid in Pieris brassicae

The digestion and absorption of dietary glycerol tri(1-¹⁴C)oleate and oleic acid-1-¹⁴C and subsequent transport of the label was followed during the fifth instar in Pieris brassicae. The rate of incorporation of the label in tissue lipid was similar in both diets. Triolein was hydrolysed to free fatty acids (FFA), diglycerides (DGL), and monoglycerides (MGL). DGL were rapidly absorbed. FFA were less readily absorbed and some were excreted. In the gut wall the label was found in phospholipids (PL), triglycerides (TGL), DGL, and FFA. In haemolymph most of the label was in DGL and PL, but later appeared also in TGL and sterol esters (SE). The results suggest that DGL are released from the gut wall and carried in haemolymph into the fat body. In the fat body lipid is stored mainly as TGL, and released as DGL, TGL, and SE. The turnover of oleate in haemolymph DGL is rapid in comparison to haemolymph SE or TGL. Synthesis of PL in gut lumen is apparent. Much of this PL is excreted but some may be absorbed.     

Upregulation of hemolymph and tissue ferritin by dietary HgCl2 in the wax moth Galleria mellonella

The effect of Hg treatment on hemolymph and tissue ferritin in the wax moth Galleria mellonella was examined by western blotting. At 48 h after feeding HgCl₂, the level of hemolymph ferritin increased approximately 1.8‐fold over that of control insects that were not fed HgCl₂, while there was a small increase in tissue ferritin. Time series experiments showed that tissue ferritin had a typically saturated pattern, with a maximum level from 24 to 72 h, although it decreased 12 h following HgCl₂ feeding, while hemolymph ferritin first decreased but subsequently increased. Tissue ferritin in the fat body, gut and Malpighian tubules, the main tissues of ferritin expression, was upregulated over time following treatment with Hg, and in particular, tissue ferritin in the gut increased by a large amount at 12–48 h. The results suggest that in G. mellonella, the ferritin‐inducible mechanisms following treatment with HgCl₂ are different for hemolymph and tissue ferritin, as are their biochemical properties.     

Nosema invadens sp. n. (Microsporida: Nosematidae), a pathogen causing inflammatory response in Lepidoptera

States in the life cycle of Nosema invadens sp. n. are described from the raisin moth, Cadra figulilella, and the almond moth, Cadra cautella. In laboratory tests, larvae of the following Lepidoptera were also susceptible: Ephestia elutella, Plodia interpunctella, Galleria mellonella, and Paramyelois transitella. The midgut wall and Malpighian tubules were invaded first, but subsequently most other tissues were also infected. Severe inflammatory response accompanied infection, including hemocytic encapsulation of infected areas and hemocytic infiltration of fat tissue.     

Uric acid metabolism during pupal-adult development of Pieris brassicae

Uric acid metabolism has been investigated during the pupal and adult stages of Pieris brassicae. Uric acid and its main metabolite, allantoic acid, have been quantified in various organs (fat body, gut, wings) during development, in order to determine synthesis, degradation, and transport phenomena. Both labelling experiments (using 2-¹⁴C uric acid, guanine, and guanosine) and enzymatic studies (xanthine dehydrogenase, guanine deaminase, and uricase) were performed.Labelled uric acid, when injected into a young pupa, accumulates preferentially into the fat body, and its degradation leads to an increase in allantoic acid, which is found chiefly in imaginal structures (wings, heads, body wall). Since uricase is present only in low levels through the pupal stage, only a small fraction of uric acid is metabolized.In the developing pharate adult, uric acid is transported via the haemolymph from fat body to the wings and gut. Male wings accumulate more uric acid than female wings. At emergence, a large amount of uric acid and most of the allantoic acid are excreted into the meconium, but not together; uric acid is excreted into the so-called ‘meconium 1’ containing ommochromes, whereas its metabolite is eliminated only after wing expansion into ‘meconium 2’, a colourless fluid. Shortly before emergence, the fat body recovers its ability to synthesize uric acid, a fraction of which is excreted within ‘meconium 1’.During adult life, the synthesis of uric acid occurs in the fat body and ovaries, where it is especially abundant. Ageing organs (wings, heads, testes) accumulate it markedly. A small fraction is excreted together with allantoic acid by the butterfly.Purine catabolism pathways have been investigated, showing that in guanine derivatives, the freebase state of guanine leads quickly to uric acid (and its metabolites), whereas ¹⁴C-guanosine may be transformed into nucleotide and incorporated efficiently into wing pteridines when it is injected at the time of adult pigmentation.Another purine derivative, identified as adenosine, has been shown to accumulate in male fat body just before adult emergence. Its amount increases during the first days of emerged adult life, and it corresponds to an alternative pathway of purine catabolism. Its absence in females is related to development of the ovaries.     

Studies on the in vivo cellular reactions and fate of injected bacteria in Galleria mellonella and Pieris brassicae larvae

Galleria mellonella and Pieris brassicae were injected with carefully standardized doses of killed Bacillus cereus and other bacteria, and blood cell and bacterial counts were made in the first hour postinjection. A very rapid falloff in hemocyte and bacterial numbers was accompanied by the formation of clumps, composed of hemocytes and bacteria, attached to the internal surfaces of the insects. The fall was followed by a rise in blood cell numbers. The magnitude of the fall increased with increasing bacterial dosage, down to a limiting value, and a theoretical model of the process was found to agree well with the data. It is suggested that clumping is a cellular defense reaction and that it is related to the production of nodules.     

Cellular defense reactions of insect hemocytes in vivo: Nodule formation and development in Galleria mellonella and Pieris brassicae larvae

Galleria mellonella and Pieris brassicae larvae were injected with a standardized dose of killed Bacillus cereus and other bacteria and the reactions of hemocytes followed in the first 24 hr by dissection and histology. Nodules formed in all insects injected with nonpathogens, but a pathogen, Staphylococcus aureus, failed to provoke this reaction. Within 5 min, clumps consisting of granular hemocytes, plasmatocytes, and bacteria were found attached to the internal surfaces of the insects. In the following hours, the cells comprising the clumps broke down and merged with a melanizing acellular substance, and the necrosing masses became encapsulated by plasmatocytes to form mature nodules. The role of granular hemocytes in the formation of the initial cell/bacteria aggregates is discussed along with the possible importance of nodules to the cellular defense reactions of insects.     

RNA synthesis in imaginal disks of Galleria mellonella: Effects of alpha- and beta-ecdysone and fat body in vitro

The effects of alpha-ecdysone (α-E), beta-ecdysone (β-E), and larval fat body on morphogenesis and total RNA synthesis in wing imaginal disks of Galleria mellonella were studied. Both ecdysones induce morphogenesis of disks in vitro. Alpha-ecdysone and β-E (0·3–3·0 μg/ml) stimulate RNA synthesis 30 and 60 per cent above control levels, respectively. While less α-E (0·3 μg/ml) is required to increase RNA synthesis than to induce morphogenesis, the reverse is true for β-E. Morphogenesis (i.e. tracheole migration and evagination) can proceed in the presence of concentrations of β-E (0·03 μg/ml) that are subthreshold for the induction of RNA synthesis (0·3 μg/ml). We conclude, therefore, that the increase in total RNA (presumbly ribosomal) is unrelated to and not a prerequisite for tracheole migration or evagination. If morphogenically active preconditioned medium (i.e. medium in which α-E and fat body have been incubated for 48 hr and the fat body then removed) is added to disk cultures, RNA synthesis is not stimulated. Apparently, increases in total RNA caused by both ecdysones may not be necessary for early in vitro disk development. The independent nature of some ecdysone-induced events and implications of our conclusions are discussed.     

Some aspects of regulation of fat body lipolytic activity in Galleria mellonella

The degradation of ¹⁴C-trioleate by the homogenate of fat body of normally developing specimens of both sexes of Galleria mellonella at different days after the larval-pupal ecdysis, and of specimens subjected to different experiments was studied. It has been confirmed that the lipolytic activity of female fat body being low after the larval-pupal ecdysis, rises distinctly by about 6 days later. In contrast to this, the lipolytic activity of this tissue in males is high and does not appear to undergo changes.Ovariotectomy causes a significant fall of lipolytic activity.Preincubation of fat body of ovariotectomized females in the medium containing ovaries of pharate adults 6 days after the larval-pupal ecdysis brings about a rise of lipolytic activity of this tissue.     

A standardizable protocol for infection of Rhodnius prolixus with Trypanosoma rangeli, which mimics natural infections and reveals physiological effects of infection upon the insect

Trypanosoma rangeli is a protozoan parasite that shares hosts - mammals and triatomines - with Trypanosoma cruzi, the etiological agent of Chagas disease. Although T. rangeli is customarily considered to be non-pathogenic to human hosts, it is able to produce pathologies in its invertebrate hosts. However, advances are hindered by a lack of standardization of infection procedures and these pathologies need documentation. To establish a suitable, and standardizable, infection protocol, the duration of the fourth instar was evaluated in nymphs infected by injection into the thorax with different concentrations of parasites, and compared with nymphs infected naturally (i.e. orally). We demonstrate that delays in moult were attributable to the presence of the parasite in the haemolymph (vs. the gut) and propose that the protocol presented here simulates closely natural infections. This methodology was then used for the evaluation of physiological parameters and several hitherto unreported effects of T. rangeli infection on Rhodnius prolixus were revealed. Haemolymph volume was greater in infected than uninfected nymphs but this alteration could not be attributed to water retention, since infected insects lost the same amount of water as controls. However, we found that lipid content and fat body weight were both increased in insects infected by T. rangeli. We propose that this is due to the parasite’s sequestration of host blood lipids and carrier proteins. With these findings, we have taken a few first steps to unravelling physiological details of the host-parasite interaction. We suggest future directions towards a fuller understanding of mechanistic and adaptive aspects of triatomine-trypanosomatid interactions.     

Yeastlike fungi from greater wax moth larvae (Galleria mellonella) fed antibiotics

The effect of the antibiotics oxytetracycline, chloramphenicol, and penicillin on the gut flora of Galleria mellonella larvae was not only to suppress Streptococcus faecalis, a typical gut organism of all stages of the moth, but also simultaneously to cause an increase in the number of yeastlike fungi, Candida guilliermondi, Candida krusei, and Geotrichum candidum. Nystatin prevented or minimized yeast multiplication. Most pupae and adults were sterile or contained only S. faecalis, even when prepupae had contained many fungi. A combination of oxytetracycline-nystatin in a total dosage of 1 and 3 × MIC/cm² of honeycomb surface, respectively, reduced both S. faecalis and fungal counts, so that after a 3-day incubation, most of the larvae were sterile or near sterile.     

Lysozymelike lytic enzyme of Streptococcus faecalis and its role in the larval development of wax moth, Galleria mellonella

The predominant type of bacteria present in the gut of larval Galleria mellonella were streptococci group D identified as Streptococcus faecalis which showed bacteriolytic activity. Young larvae usually contained mixed populations with a marked dominance of fecal streptococci while normally developed mature larvae most frequently contained large uniform populations of S. faecalis. Pupal stages were found to contain the highest percentage of individuals with pure cultures of fecal streptococci.The author suggests a hypothesis that, owing to its bacteriolytic properties, S. faecalis can be considered as a component of the natural, nonspecific defense mechanism of G. mellonella against bacterial infections. The lytic enzyme released in the exponential growth phase of S. faecalis participates in the selection process stabilizing the microbial flora of wax moth larvae; it limits the population of other forms of bacteria. Larval resistance to bacterial infections to a large extent depends on the magnitude of the populations and thus on S. faecalis muramidase concentration. Bacterial lysozyme inhibited the growth of the ingested organisms and in consequence it prevented the proliferation of undesired bacteria in the digestive tract of Galleria larvae.The lytic enzyme proved to be identical with autolysin, a β-N-acetylmuramide glycanhydrolase (EC 3.2.1.17) which has been isolated from trypsin-speeded wall autolysates of S. faecalis by Shockman and Cheney (1969).     

Changes in body tissues and hemolymph composition of Culex pipiens in response to infection by Romanomermis culicivorax

Hemolymph composition of fourth instar larvae of an autogenous strain of Culex pipiens was examined to determine the effects of parasitism by a mermithid nematode, Romanomermis culicivorax. Mosquitoes were reared under two different pH regimens: 4.5 and 7.3. Wet and dry weight of infected mosquitoes reared at either pH were significantly lower than controls. The effects of parasitism in the development of C. pipiens were evaluated from paraffin sections of mosquito larvae 2, 4, and 6 days postinfection. At 2 days postinfection, the infected larvae showed no apparent effects of parasitism; at day 4, the fat body tissue was reduced and imaginal disc development was retarded; and at day 6, parasitized mosquitoes were smaller in cross section, fat body tissue was found only in isolated clumps, and there was a complete absence of imaginal discs. Concentrations of total carbohydrates in hemolymph from infected fourth instar mosquitoes reared at pH 7.3 were reduced. Trehalose and glucose were each reduced by more than half. Total α-amino nitrogen was significantly lower in infected mosquitoes reared at pH 7.3. However, total amino acid concentrations for hemolymph from control and infected larvae reared at pH 7.3 were the same. Methionine sulfoxide decreased 63% and proline increased 2.5 times in infected mosquitoes. Hemolymph protein concentrations were reduced 80% in infected mosquitoes reared at both pHs. The number of hemolymph proteins also declined from 35 to 22 during infection. Two host proteins, 82,000 and 158,000 daltons, remained prominent throughout the mermithid infection.     

A recombinant immunosuppressive protein from Pimpla hypochondriaca (rVPr1) increases the susceptibility of Lacanobia oleracea and Mamestra brassicae larvae to Bacillus thuringiensis

The precise mechanisms underlying Bacillus thuringiensis-mediated killing of pest insects are not clear. In some cases, death may be due to septicaemia caused by Bt and/or gut bacteria gaining access to the insect haemocoel. Since insects protect themselves from microbes using an array of cellular and humoral immune defences, we aimed to determine if a recombinant immunosuppressive wasp venom protein (rVPr1) could increase the susceptibility of two pest Lepidoptera (Lacanobia oleracea and Mamestra brassicae) to Bt. Bio-assays indicated that injection of 6 μl of rVPr1 into the haemocoel of both larvae caused similar levels of mortality (less than 38%). On the other hand, the LD_30-40 of Bt for M. brassicae larvae was approximately 20 times higher than that for L. oleracea larvae. Furthermore, in bio-assays where larvae were injected with rVPr1, then fed Bt, a significant reduction in survival of larvae for both species occurred compared to each treatment on its own (P < 0.001); and for L. oleracea larvae, this effect was more than additive. The results are discussed within the context of insect immunity and protection against Bt.     

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.