Adipokinetic hormone-induced lipid mobilization and lipophorin interconversions in fifth larval instar locusts

The response of fifth larval instar locusts to injected adipokinetic hormone (AKH) is only poor, as is reflected in both a very moderate elevation of the haemolymph lipid concentration and the slight occurrence of the haemolymph lipophorin interconversions characteristic for adult locusts, resulting in formation of only small quantities of the low density lipophorin (A⁺). However, an additional lipophorin fraction (A′) is induced, which is intermediate in density and size between high and low density lipophorin and which is not identified in adult haemolymph. As in adults, larval A⁺ formation includes association of the resting high density lipophorin with a non-lipid containing protein (C₂), the haemolymph concentration of which is only one-fifth relative to adults. However, the larval haemolymph protein composition is not the primary cause of the incomplete adipokinetic response, as elevation of the concentration of protein C₂ by injection of isolated adult C₂, whether or not in combination with adult high density lipophorin, did not increase lipophorin conversions nor haemolymph lipid elevation.In vitro incubation of larval fat bodies in adult haemolymph showed that competency to both the AKH-induced lipid release and the haemolymph lipophorin conversions of the larval fat body are reduced compared to equal amounts of adult tissue. Reciprocal incubation of adult fat body in larval haemolymph resulted in only a very moderate adipokinetic response, demonstrating that larval haemolymph protein composition is restrictive for full development of hormone action.Both immunoblotting experiments and enzyme-linked immunosorbent assays (ELISA), using monoclonal antibodies specific for the adult lipophorin apoproteins, indicated that the larval lipophorins closely resemble the adult forms. Apparently the structure of locust lipophorins is remarkably constant throughout development despite changes in metabolic functions.     

3-Decanol in the haemolymph of the hermit crab Clibanarius vittatus signals shell availability to conspecifics

Hermit crabs with poor fitting shells are chemically attracted to dying gastropods and conspecifics where a shell may become available. For land hermit crabs, the shell cue is a volatile compound found in the haemolymph. Based on this knowledge, we tested the hypothesis that shell investigation behavior in aquatic hermit crabs, the ancestral predecessors of terrestrial hermit crabs, is also triggered by volatile cues. Volatile compounds from haemolymph of Clibanarius vittatus and Pagurus pollicaris and brachyuran decapod crustaceans were purged from a water-haemolymph solution, trapped in seawater and tested for induction of shell investigation behavior with juvenile C. vittatus. Only volatiles from C. vittatus haemolymph stimulated shell investigation. Volatile compounds were isolated from haemolymph by headspace solid-phase microextraction (SPME) and analyzed by coupled gas chromatography-mass spectrometry (GC-MS). Two prominent compounds were identified, 3-decanol, which was unique to C. vittatus haemolymph, and 2-ethyl-1-hexanol, which was present in the haemolymph of all 4 crustacean species. In shell investigation bioassays, 3-decanol from C. vittatus haemolymph stimulated shell investigation behavior, while 2-ethyl-1-hexanol did not. In bioassays with synthetic 1-, 2-, 4-, and 5-decanol, shell investigation behavior was evoked by 1-decanol, 5-decanol and 3-undecanol. There was no response to 2- and 4-decanol. The response of C. vittatus to volatile shell cues supports the hypothesis that volatile cue detection evolved prior to the occupation of terrestrial niches by crustaceans.     

Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens

In Locusta migratoria, activation of phenoloxidase in the haemolymph in response to injection of laminarin is age-dependent: being absent in fifth instar nymphs and newly emerged adults, and only becoming evident four days after the final moult. This pattern of change in phenoloxidase activation correlates with the pattern of change in the concentration of apolipophorin-III (apoLp-III) in the haemolymph. Injection of a conspecific adipokinetic hormone (Lom-AKH-I) has no effect on the phenoloxidase response in nymphs or newly emerged adults but, in adults older than four days, co-injection of the hormone with laminarin prolongs the activation of phenoloxidase in the haemolymph: a similar enhancement of the response to laminarin is observed in locusts that have been starved for 48 h but not injected with AKH-I. During most of the fifth stadium, injection of laminarin results in a decrease in the level of prophenoloxidase in the haemolymph; an effect that is not observed in adults of any age. Marked changes in the concentration of apoLp-III, and the formation of LDLp in the haemolymph, are observed after injection of laminarin (or LPS) and these are remarkably similar, at least qualitatively, to those that occur after injection of AKH-I. The involvement of lipophorins in the activation of locust prophenoloxidase in response to immunogens is discussed.     

昆虫血淋巴的收集技术与方法

血淋巴是昆虫生理生化研究的重要材料之一。文章分析归纳各类昆虫血淋巴的不同收集方法、收集前的昆虫准备以及收集后血淋巴的预处理,并结合该试验室研究阐述在收集昆虫血淋巴过程中的一些问题和经验,为能有效提取血淋巴提供一定的方法支持。     

Differences in adipokinetic response of Pyrrhocoris apterus (Heteroptera) in relation to wing dimorphism and diapause

Three experimental groups of adult females (reproductive and diapausing brachypters, and macropters with reproductive arrest) of Pyrrhocoris apterus (Linnaeus) (Heteroptera: Pyrrhocoridae) from a temperate population were analysed for their adipokinetic responses. The adipokinetic response, expressed as an increase of haemolymph lipids after injection of adipokinetic hormone from Locusta migratoria (Lom-AKH-I), was assessed in relation to age, wing dimorphism and type of reproductive arrest. Two pmols of Lom-AKH-I were used for determination of adipokinetic responses. The increase of haemolymph lipids in all experimental groups of females induced by this dose of the hormone was comparable with that induced by crude extract of the bug’s own corpora cardiaca. The level of adipokinetic response after injection of 2 pmol of Lom-AKH-I was significantly higher in macropterous and diapausing brachypterous females than in reproductive brachypterous females. However, significantly higher contents of haemolymph lipids in control macropterous females than those found in the control reproductive and diapausing brachypterous females of the corresponding age revealed wing-morph-related differences in lipid metabolism. The observed wing morph- and diapause-related differences in the content of haemolymph lipids and adipokinetic response, respectively, are discussed in relation to the different roles of two wing morphs in the life history of this heteropteran.     

Experimental infection of ixodid ticks with Karshi virus

The ixodid ticks Hyalomma asiaticum, H. anatolicum, Dermacentor niveus were infected experimentally with Karsha virus. The virus replication has been proved to occur in the tick's organism. The titre of the virus grows gradually in infected ticks. Entering the tick's gut during its feeding virus particles penetrate into the gut walls where primary multiplication and accumulation of the virus take place.     

杀虫环对昆虫血淋巴糖和游离氨基酸的影响

 用毛细管色谱和离子交换色谱法分析了黑胸大蠊雄性成虫血淋巴糖和游离氨基酸的组分、合量及杀虫环对它们的影响,并与电生理记录的EPSP电位比较,表明:1.黑胸大蠊血淋巴有五种糖,海藻糖、葡萄糖含量最多。2×10~(-5)mol/L杀虫环作用于虫体5分钟后,五种糖含量迅速增加;30分钟后下降23～47％。2.血淋巴中含有16种游离氨基酸及大量的NH_3。杀虫环可降低14种氨基酸的含量,尤其对生糖氨基酸(丙、异亮、酪、脯)的影响显著。3.2×10~(-5)mol/L杀虫环作用5分钟可增加中枢第Ⅵ腹神经节自发电位的释放频率和振幅,6～7分钟后迅速减少并消失,此时诱发EPSP电位振幅下降。18～20分钟突触传递被阻断。此过程恰巧与血淋巴糖代谢的加快而减缓的过程一致。     

Anaerobic capacity of a crustacean sensitive to low environmental oxygen tensions,the freshwater amphipod Gammarus pulex (L.)

We investigated the accumulation of haemolymph L-lactate in response to anoxia, progressive hypoxia and activity, in a species that rarely encounters low PO₂ in its natural environment, the freshwater amphipod Gammarus pulex (L.). Individuals survived <1.5 h anoxia and accumulated L-lactate in the haemolymph at a rate of 10.26 mmol l^–1 h^–1. No lactate was excreted into the medium during this time. Despite the fact that most individuals of G. pulex were oxyconformers, exposure to acutely declining PO₂s did not result in a switch to anaerobic pathways until PO₂ < 4.4 kPa. Even then the concentrations accumulated remained very low until PO₂ < 0.46 kPa. There was no accumulation of L-lactate in the haemolymph as a result of exhaustive activity. Consequently, it was suggested that (a) it cannot be automatically assumed that anaerobic metabolism is switched on only at the point at which oxyregulation breaks down, (b) that the poor anaerobic capacity observed plays little, if any, role in the metabolic response to hypoxia by Gammarus pulex.     

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。     

Influence of fungal infection on the DOPA-semiquinone and DOPA-quinone production in haemolymph of Galleriamellonella larvae

The formation of reactive oxygen metabolites in haemolymph of Galleria mellonella larvae was studied by ESR spectroscopy. The inhibition of the production of the reactive oxygen metabolites of DOPA in haemolymph under the action of fungal infection was shown using spin trap 1-hydroxy-3-carboxy-pyrrolidine. This inhibition correlated with decrease of phenoloxidase activity in haemolymph of infected insects. Simultaneously, the decrease of production of DOPA-semiquinone was detected using method of spin stabilization by diamagnetic metal ions. Moreover, it was shown that the formation of DOPA-quinone was slowed down in haemolymph of infected insects. Our results suggest that the DOPA-derived quinones/semiquinones may be involved in immune response of insects as part of its defense mechanism.     

Investment in immune function under chronic and acute immune challenge in an insect

In this paper we investigate the relationship between the chronic burden of mid‐gut parasites (eugregarine trophozooites) and the effect of an acute haemolymph challenge (a nylon insert) on two important insect immune effector systems (phenol oxidase (PO) and the encapsulation response) in a field‐population of damselflies. PO levels in the haemolymph, and the magnitude of the encapsulation response were maintained, regardless of chronic and subsequent acute experimental immune challenges. The maintenance of these effector systems is therefore probably an important life‐history requirement in these damselflies. Investment in mid‐gut PO levels was significantly negatively related to the animal's chronic parasite burden after an acute experimental challenge in the haemolymph, suggesting that maintaining PO levels across two physiological compartments (haemolymph and mid‐gut) is costly. The results suggest that the immune effector system activity in different physiological compartments in an insect's body is affected by chronic parasite burdens in the face of the demands imposed by an acute immune insult.     

Physical stress primes the immune response of Galleria mellonella larvae to infection by Candida albicans

Larvae of the greater wax moth (Galleria mellonella) that had been subjected to physical stress by shaking in cupped hands for 2 min showed reduced susceptibility to infection by Candida albicans when infected 24 h after the stress event. Physically stressed larvae demonstrated an increase in haemocyte density and elevated mRNA levels of galiomicin and an inducible metalloproteinase inhibitor (IMPI) but not transferrin or gallerimycin. In contrast, previous work has demonstrated that microbial priming of larvae resulted in the induction of all four genes. Examination of the expression of proteins in the insect haemolymph using 2D electrophoresis and MALDI TOF analysis revealed an increase in the intensity of a number of peptides showing some similarities with proteins associated with the insect immune response to infection. This study demonstrates that non-lethal physical stress primes the immune response of G. mellonella and this is mediated by elevated haemocyte numbers, increased mRNA levels of genes coding for two antimicrobial peptides and the appearance of novel peptides in the haemolymph. This work demonstrates that physical priming increases the insect immune response but the mechanism of this priming is different to that induced by low level exposure to microbial pathogens.     

桑叶营养对家蚕血淋巴阳离子水平的调节作用

调查了取食不同品种桑树（M-5, S-36和V-1）叶片的家蚕（多化性品种Pure Mysore、 二化性品种NB4D2和CSR2）5龄幼虫血淋巴中阳离子的变化。结果表明: 家蚕幼虫血淋巴中Na+浓度低, K+和Mg2+浓度很高, Ca2+浓度较高。在幼虫活跃取食期间, 血淋巴中阳离子水平显著提高。血淋巴中阳离子水平与排泄物的量呈负相关。血淋巴中阳离子水平受阳离子外泌的控制。与非取食阶段相比, 取食阶段的阳离子清除速率低。家蚕二化性品种的血淋巴阳离子水平比多化性品种PM高出30%。结果说明家蚕品种对家蚕血淋巴阳离子变化的影响显著大于桑树品种或家蚕个体发育的影响。     

Modelling the subgenual organ of the honeybee, Apis mellifera

In a recent study on the honeybee (Apis mellifera), the subgenual organ was observed moving inside the leg during sinusoidal vibrations of the leg (Kilpinen and Storm 1997). The subgenual organ of the honeybee is suspended in a haemolymph channel in the tibia of each leg. When the leg accelerates, the inertia causes the haemolymph and the subgenual organ to lag behind the movement of the rest of the leg. To elucidate the biophysics of the subgenual organ system of the honeybee, two mathematical models to simulate the experimentally observed mechanical response are considered. The models are a classical mass-spring model and a newly developed tube model consisting of an open-ended, fluid-filled tube occluded by an elastic structure midway. Both models suggest that the subgenual organ included in the haemolymph channel resembles that of an overdamped system. In resembling the biophysics of the subgenual organ system in the honeybee, we consider the tube model to be the better of the two because it simulates a mechanical response which complies best with the experimental data, and the physical parameters in the model can be related to the␣constituent parts of the subgenual organ included in the haemolymph channel. Received: 25 July 1997 / Accepted in revised form: 8 December 1997     

Examining the relationship between hemolymph phenoloxidase and resistance to a DNA virus, Plodia interpunctella granulosis virus (PiGV)

We have a detailed understanding of invertebrate immune responses to bacteria and fungal pathogens, but we know less about how insects respond to virus challenge. Phenoloxidase (PO) functions as an important immune response against many parasites and pathogens and is routinely used as a measure of immune competance. We examine the role of haemolymph PO activity in Plodia interpuncetella's response to its natural granulosis virus (PiGV). Larvae were challenged with virus by both oral inoculation of occluded virus (the natural infection route) and direct intrahaemocoelic injection of budded virus. Haemolymph was collected at time points post-viral challenge using a novel method that allows the volume of haemolymph to be quanitified. The haemolmyph was collected without killing the larvae so that haemolymph samples from individuals that developed viral disease could be distinguished from samples collected from those that fought off infection. The level of haemolymph PO activity in resistant larvae did not differ from control larvae. Therefore we have no evidence that PO is involved in resistance to virus in the haemocoel whether larvae are challenged naturally by oral innoculation or directly by intraheamocoelic injection. Phenoloxidase may therefore not be a relevant metric of immunocompetence for viral infection.     

Effect of repeated sampling on haemolymph pH,PO2 and haemocyte activity in the Pacific oyster,Crassostrea gigas (Thunberg)

The effects of repeat bleeding via a novel cannulation technique, on the physiology of the Pacific oyster, Crassostrea gigas (Thunberg), were compared to control animals from which haemolymph was sampled by syringe and needle. The effects of surgery and cannulation were characterized by an initial alkalosis (up to 8 h) in the haemolymph of cannulated animals followed by an acidosis of up to 12 h; which occurred in both cannulated and control oysters. A decline in partial pressure of oxygen of oyster haemolymph, observed in both cannulated and control oysters, was directly related to shell closure following handling. Furthermore, a haemocyte concentration observed at 4 h followed by haemocyte dilution at 12 h post-surgery, suggested a handling-related stress response in both cannulated and control oysters. A heightened phagocytic activity of haemocytes at 4 h also supported the occurrence of handling-related stress response in all animals. The cannulation technique described here provides the investigator with a convenient tool for chronic, and repeated sampling of haemolymph with a transient disturbance to the animal.     

The toxic effects of phenoloxidase from the haemolymph of tenebrionid beetles

The injection of haemolymph originating from several species of tenebrionid beetles into blowfly larvae caused a gradual paralysis accompanied by colour changes in the haemolymph of the injected test insects. It was found that the lethal effect of the haemolymph of the beetle Blaps sulcata was due to phenoloxidase. The enzyme was activated by the exposure and incubation of the haemolymph at room temperature.The identity between the toxic factor and phenoloxidase in the beetle's haemolymph was demonstrated by the following data: (1) A correlation between the rate of lethal and phenoloxidase activities during the activation process of the toxic haemolymph. (2) Phenylthiourea, a well-known inhibitor of phenoloxidase, inhibited both the enzymatic and the toxic action of the beetle's haemolymph. (3) A commercial preparation of phenoloxidase (originating from mushrooms) imitated the lethal effects and the accompanying symptoms of the toxic haemolymph. (4) Sephadex G-100 column separation of the Blaps haemolymph revealed a complete overlap between the enzymatic and lethal regions of the elution pattern.The possible effects of phenoloxidase on the haemolymph of the injected insects are discussed.     

Immune responses of the argasid tick Ornithodoros moubata moubata induced by infection with the filarial worm Acanthocheilonema viteae

Investigations were undertaken to determine whether the tick Ornithodoros moubata moubata mounted a detectable immune response to primary and secondary infections with Acanthocheilonema viteae. Uninfected control tick survival rate was 70%, but only 45% in the primary infection group. Post-secondary infection survival rate (82%) was comparable to controls, indicating that these selected ticks had some protective advantage. Mean A. viteae infective larvae recovery from ticks with secondary infections was 31.4% lower than expected, suggesting the development of immunity. SDS-PAGE of haemolymph for proteins induced post-primary infection yielded a stronger signal at 45 kDa than controls, which was further elevated post-secondary infection. Proteins at 48, 22 and 16 to 18 kDa were detected in haemolymph from infected ticks but not seen from controls. The direct effect of haemolymph on microfilarial viability was examined using a novel in vitro assay; in these preliminary trials no differences were observed in parasite viability when exposed to haemolymph from infected or uninfected groups of ticks.     

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.     

Heritability of immune function in the caterpillar Spodoptera littoralis

Phenoloxidase (PO) is believed to be a key mediator of immune function in insects and has been implicated both in non-self recognition and in resistance to a variety of parasites and pathogens, including baculoviruses and parasitoids. Using larvae of the Egyptian cotton leafworm, Spodoptera littoralis, we found that despite its apparent importance, haemolymph PO activity varied markedly between individuals, even amongst insects reared under apparently identical conditions. Sib-analysis methods were used to determine whether individuals varied genetically in their PO activity, and hence in one aspect of immune function. The heritability estimate of haemolymph PO activity was high (h(2) = 0.690 +/- 0.069), and PO activity in the haemolymph was strongly correlated with PO activity in both the cuticle and midgut; the sites of entry for most parasites and pathogens. Haemolymph PO activity was also strongly correlated with the degree to which a synthetic parasite (a small piece of nylon monofilament) was encapsulated and melanized (r = 0.622 +/- 0.142), suggesting that the encapsulation response is also heritable. The mechanism maintaining this genetic variation has yet to be elucidated.