Possible involvement of an enzymatic system for superoxide generation in lepidopteran larval haemolymph

The superoxide generative system in Pseudaletia separata larval haemolymph plasma was shown to consist of at least two components, low (LMF) and high (HMF) molecular weight factors using ultrafiltration, dialysis, and gel filtration. The LMF was concluded to be a molecule(s) smaller than 5 kDa while the HMF to be a protein(s) larger than 100 kDa. The total amount of superoxide produced depended on the amount of LMF and was independent of that of HMF added to the reaction mixture. Both the LMF and HMF were required for O₂^? generation. From results in the present article, it was hypothesized that the LMF was a substrate(s) discharging electrons and HMF was an enzyme(s) to mediate the electron transfer to O₂ forming O₂^?. Superoxide production was detected in the haemolymph plasma of 5 lepidopteran species in addition to P. separata. It was concluded that superoxide production is a common phenomenon at least in these lepidopterans. © 1995 Wiley-Liss, Inc.     

Changes in haemolymph octopamine levels associated with food deprivation in the locust, Schistocerca gregaria

ABSTRACT. Levels of octopamine in the haemolymph and locomotor activity have been measured over 24 h in fed and food-deprived adult male locusts, Schistocerca gregaria (Forskål). Locusts are predominantly diurnal, the locomotor activity of individuals with continuous access to food was higher during the photophase. The basal level of octopamine in haemolymph sampled during the first quarter of the light cycle was 7.8±1.1 pg/μl (51 nm). Depriving locusts of food at the start of the photophase for 9 h caused an increase in both the speed of movement and total activity compared to fed individuals. There was a corresponding increase in the haemolymph octopamine concentration which continued for as long as the insects were unable to feed, reaching a concentration of 20.7 ± 3.7 pg/μl (135 nM). While declining, the levels of octopamine remained higher in fed locusts for up to 4 h after replacing the food. The results are discussed in relation to the regulation of feeding behaviour and a proposed neurohormonal role of octopamine in insects.     

Major haemolymph proteins inCeratitis capitata: Biosynthesis and secretion during development

Summary The accumulation of major haemolymph proteins (a group of proteins immunologically related to Calliphorin) their biosynthesis in vivo and in organ culture as well as their secretion, has been studied during the late larval stages and white pupae of the Mediterranean fruit flyCeratitis capitata. The accumulation of major haemolymph proteins in the haemolymph, shows a twenty fold increase from the 4-day old larvae to the white pupae stage, while in the fat body there is only a seven fold increase. It is evident from the in vivo and organ culture studies, that the major haemolymph proteins are synthesized during the late larval stage and their synthesis declines abruptly during the stage of white pupae. It seems also that each polypeptide has its own characteristic developmental kinetics of synthesis. The major haemolymph proteins are synthesized in the fat body and are very quickly secreted into the haemolymph.     

Protein release from the larval fat body of the southwestern corn borer, Diatraea grandiosella: An in vitro study

The release of protein from the perivisceral fat body of non-diapausing, pre-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Time course studies showed a selective release of proteins into macromolecule-free Grace's medium. The rate of release of individual proteins differed. The release of some proteins was partially inhibited by the incorporation of potassium cyanide (10^?2 M) and ouabain (5 × 10^?3 M) into the medium. During a 5 min incubation a single major high molecular weight protein fraction was released at a high rate from the fat body of both non-diapausing and diapausing larvae. A low molecular weight protein (the diapause-associated protein) was also released readily from the fat body of diapausing larvae. Although most proteins released from the fat body in vitro appeared to be present in the haemolymph in vivo, one notable exception was the absence of the diapause-associated protein from the haemolymph. The method holds promise for facilitating further studies of protein release from insect fat body.     

Bracken, ants and extrafloral nectaries. III. How insect herbivores avoid ant predation

Abstract. 1. Ants (Myrmica spp. and Formica lemani) visiting the extrafloral nectaries of bracken, Pteridium aquilinum, imposed heavy mortality on caterpillars of a novel moth species experimentally introduced onto bracken fronds. However, the exclusion of ants from bracken fronds had no significant effect on adapted bracken-feeding herbivores in Britain.
2. The feeding stages of British bracken-feeding insects are immune to, or can largely avoid, predation by ants in a variety of ways. Gall formers and miners cannot be attacked by these ants. Two other species hide, one inside tied leaves, the other in a mass of 'spittle'. Another group of species jumps away from, or falls off the plant when touched by ants. Sawfly caterpillars in the genera Strongylogaster, Aneugmenus and Tenthredo have viscous, distasteful haemolymph that repels ants.
3. No species of bracken herbivore has an absolute temporal refuge from ants; during their development they all overlap with ants to some degree.
4. Ant predation appears to have played a significant role in determining the contemporary structure of British bracken-feeding insect communities. Distasteful haemolymph in sawfly caterpillars may have evolved in response to selection from ant predation. Other species may fortuitously possess characteristics, evolved in response to a variety of selective forces, that also reduce the impact of ants; without such characteristics, however, we postulate that they would be unable to live on this plant. An absence of external, foliage feeding Lepidoptera early in the spring, a high proportion of sawfly species, and a high proportion of gall-formers and miners may all be characteristics of the bracken herbivore community which have been influenced by ant predation.     

Expression of a cystine-rich fish antifreeze in transgenicDrosophila melanogaster

We have usedDrosophila melanogaster as a model system for the transgenic expression of cystine-rich Type II antifreeze protein (AFP) from sea raven. This protein was synthesized and secreted into fly haemolymph where it migrated as a larger species (16 kDa) than the mature form of the protein (14 kDa) as judged by immunoblotting.Drosophila-produced Type II AFP demonstrated antifreeze activity both in terms of thermal hysteresis (0.13 °C) and inhibition of ice recrystallization. Recombinant AFP was purified and N-terminal sequencing revealed a 17 aa extension that began at the predicted signal peptide cleavage point. The expression of all three AFP types in transgenicDrosophila has now been achieved. We conclude that the globular Type II and Type III AFPs are better choices for antifreeze transfer to other organisms than is the more widely used linear Type I AFP.     

Étude préliminaire des variations circadiennes des protéines de l'hémolymphe de Penaeus japonicus Bate

The protein contents of the haemolymph in the decapod crustacean Penaeus japonicus Bate exhibit a tricircadian rhythm characterized by two maxima during the night and another one during the day. During the night the total protein concentration in haemolymph is higher in males. Maximal values in males are approximately four times greater (100 mg/ml) than minimal ones (25 mg/ml). Variations are less in females: maximal values are approximately two and a half times higher (75 mg/ml) than minimal ones (30 mg/ml).Electrophoresis in polyacrylamide gradient showed that the different protein constituents in general exhibit similar circadian variations. Time of acrophases depends on the sex and the stage in the intermoult cycle. On the other hand, the mean concentration of each protein constituent calculated from values measured at different times of the day are similar whatever the sex and moulting stage. Between 0 and 6 h certain protein fractions - two in females and one in males - are no longer detectable by electrophoresis. At 12 h, concentration of haemocyanin and its subunits increase simultaneously in both sexes.     

Effects of methoprene,a juvenile hormone analogue,on the larval and pupal stages of the yellow fever mosquito, Aedes aegypti

Exposure of early fourth-instar larvae of Aedes aegypti to the juvenile hormone analogue Altosid ZR15^® (methoprene) significantly increased the concentration of carbohydrates in the haemolymph of late fourth-instar larvae and reduced the haemolymph carbohydrate concentration of 24-h-old pupae relative to controls. Such treatment also effected a decline in haemolymph amino nitrogen levels of the pupal stage and a depletion of haemolymph proteins in late fourth-instar larvae as well as pupae. Two of nine protein fractions in the haemolymph of larvae were significantly depleted following methoprene treatment. Fourteen soluble protein fractions were present in the haemolymph of control pupae; two of these were missing from the pupae which were treated as larvae with methoprene. A further protein fraction, common to the haemolymph of both treated and control pupae, was significantly reduced in concentration as a consequence of exposure to methoprene. The juvenile hormone analogue impaired the capacity of the fat bodies of late fourth-instar larvae and pupae to synthesise proteins, resulting in a lowered concentration of fat body proteins. Glycogen levels in the fat bodies of treated larvae were significantly lower than in controls and glycogenolysis was suppressed due to an overall depletion of glycogen phosphorylase and, in pupae, a lowered ratio of active: inactive enzyme. The data are consistent with the proposition that the juvenile hormone analogue elicits neuroendocrinological changes in the target insect.     

家蚕血淋巴体外黑化观察及相关黑色素合成催化酶基因

观察不同生长期家蚕幼虫血淋巴在体外的黑化速度和对大肠杆菌生长的影响结果显示,随食桑生长幼虫血淋巴的黑化速度逐渐变快,对大肠杆菌生长的抑制作用逐渐增强。RT-PCR实验显示,黑色素合成催化酶Bm Tan、Bm Po-1、Bm Yellow-f和Bm Ddc等的基因在家蚕5 L 3 d血淋巴中表达量高,Bm Black、Bm Yellow和Bm Pah等的基因也有明显表达。q PCR分析显示,黑化病蚕中Bmtan、Bmddc、Bmyellow、Bmebony和Bmblack,尤其Bmddc表达发生了显著上调。与对照相比,Ddc酶的抑制剂能显著抑制脂多糖对血淋巴的诱导黑化作用。用大肠杆菌注射家蚕幼虫,血淋巴中多巴和多巴胺的含量明显上升。这些表明家蚕幼虫血淋巴黑化与防御免疫有关,Bmddc很可能在幼虫血淋巴的免疫黑化中发挥作用。