Anti-prion activity found in beetle grub hemolymph of Trypoxylus dichotomus septentrionalis

No remedies for prion disease have been established, and the conversion of normal to abnormal prion protein, a key event in prion disease, is still unclear. Here we found that substances in beetle grub hemolymph, after they were browned by aging for a month or heating for hours, reduced abnormal prion protein (PrP) levels in RML prion-infected cells. Active anti-prion components in the hemolymph were resistant to protease treatment and had molecular weights larger than 100 kDa. Aminoguanidine treatment of the hemolymph abolished its anti-prion activity, suggesting that Maillard reaction products are enrolled in the activity against the RML prion. However, levels of abnormal PrP in RML prion-infected cells were not decreased by incubation with the Maillard reaction products formed by amino acids or bovine serum albumin. The anti-prion components in the hemolymph modified neither cellular or cell-surface PrP levels nor lipid raft or autophagosome levels. The anti-prion activity was not observed in cells infected with 22 L prion or Fukuoka-1 prion, suggesting the anti-prion action is prion strain-dependent. Although the active components of the hemolymph need to be further evaluated, the present findings imply that certain specific chemical structures in the hemolymph, but not chemical structures common to all Maillard reaction products, are involved in RML prion formation or turnover, without modifying normal PrP expression. The anti-prion components in the hemolymph are a new tool for elucidating strain-dependent prion biology.     

Nitric oxide production in blowfly hemolymph after yeast inoculation

Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses that include both cellular and humoral components. Cellular responses are mediated by hemocytes and humoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In this work, we determined NO production in Chrysomya megacephala hemolymph and hemocytes after yeast inoculation. Assays were performed with non-infected controls (NIL), saline-injected larvae (SIL) or larvae injected with Saccharomyces cerevisiae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24 or 48h post-injection. NO levels in SIL were comparable to those measured in NIL until 12h, which might be considered the basal production, increasing at 24 and 48h post-injection, probably in response to the increased larval fragility after cuticle rupture. YIL exhibited significantly higher levels of NO than were found in other groups, peaking at 24h. l-NAME and EDTA caused a significant reduction of NO production in YIL at this time, suggesting the activity of a Ca(2+)-dependent NOS. Plasmatocytes and granular cells phagocytosed the yeasts. Plasmatocytes initiated the nodule formation and granular cells were the only hemocyte type to produce NO. These results permit us to conclude that yeasts induced augmented NO production in C. megacephala hemolymph and granular cells are the hemocyte type involved with the generation of this molecule.     

Hindgut innate immunity and regulation of fecal microbiota through melanization in insects

Many insects eat the green leaves of plants but excrete black feces in an as yet unknown mechanism. Insects cannot avoid ingesting pathogens with food that will be specifically detected by the midgut immune system. However, just as in mammals, many pathogens can still escape the insect midgut immune system and arrive in the hindgut, where they are excreted out with the feces. Here we show that the melanization of hindgut content induced by prophenoloxidase, a key enzyme that induces the production of melanin around invaders and at wound sites, is the last line of immune defense to clear bacteria before feces excretion. We used the silkworm Bombyx mori as a model and found that prophenoloxidase produced by hindgut cells is secreted into the hindgut contents. Several experiments were done to clearly demonstrate that the blackening of the insect feces was due to activated phenoloxidase, which served to regulate the number of bacteria in the hindgut. Our analysis of the silkworm hindgut prophenoloxidase discloses the natural secret of why the phytophagous insect feces is black and provides insight into hindgut innate immunity, which is still rather unclear in mammals.     

Feeding deterrent response of corn insects to β-exotoxin of Bacillus thuringiensis

Exotoxins produced by three varieties of Bacillus thuringiensis were added at different concentrations to the diets of the black cutworm, the fall armyworm, the European corn borer, and the house fly. By day 7 of treatment, mortality of the three lepidopterans was higher at the lower concentrations of exotoxin than at the higher concentrations tested; by day 14 of treatment, mortality was 90% of greater at all the exotoxin concentrations tested. In the house fly tests, mortality increased with increasing exotoxin concentration. Additional lepidopteran tests were run to study both the anomalous effect of toxin concentration on mortality and the effect of feeding inhibition evident by day 7 but not day 14 in treatments with high concentrations of the toxins. The tests, run with the European corn borer and with β-exotoxin calcium salt, showed that both effects could be attributed to a “feeding deterrent” associated with the toxin, and that the deterrent was not odoriforous and did not degrade over time. Apparently, the insects ate very little of the diets high in exotoxin, stopping quickly upon receiving a high dosage of the deterrent, but, nevertheless, having consumed enough toxin to be killed over time (between 7 and 14 days).     

Enantiospecific synthesis and insect feeding activity of sulfur-containing cyclitols

The first syntheses of two deoxythiocyanocyclitols (4-deoxy-4-thiocyano-l-chiro-inositol and deoxythiocyanoconduritol F) and two deoxysulfonylcyclitol acetals are reported by a chemoenzymatic enantioselective route. The compounds were prepared by a sequence of enzymatic and ruthenium-catalyzed dihydroxylations, and the results were studied regarding reaction conditions and co-catalyst for different derivatives. The new compounds were included in a minilibrary of deoxygenated cyclitols and evaluated for their capacity to influence the feeding behavior of Epilachna paenulata (Coleoptera: Coccinellidae), a common pest of the Curcubitaceae crops.     

Effect of metamorphosis on the major hemolymph proteins of the silkworm

During the metamorphosis of the silkworm, Bombyx mori, three major hemolymph proteins (MHPs) (molecular weights 17,000, 25,000, 27,000) were detected and found to be distributed in the hemolymph and in the tissues of several organs, such as the fat body, midgut, ovary, testis, and even eggs. The MHPs in eggs gradually decreased and disappeared during embryogenesis. The formation, distribution, and utilization of MHPs in tissues other than the gonad, however, were not affected by sex. Radioisotope experiments in vivo revealed that the MHPs were synthesized at an early period of the fifth larval instar. The synthesis of at least two of them occurred in the fat body. MHPs in the hemolymph entered the tissues at the onset of the larval-pupal transformation. On the basis of their appearance, distribution, and depletion, the MHPs may be classified as reserve proteins which are synthesized in the larval stage and utilized later in the developmental stages.     

褐边绿刺蛾的取食行为和取食量

观察褐边绿刺蛾Parasa consocia Walker2个世代幼虫取食杨树叶片的习性。结果表明,第1代幼虫7龄,幼虫期平均取食量167.7cm2/头,取食持续时间26d;第2代幼虫8龄,幼虫期平均取食量170.5cm2/头,取食持续时间27d。2个世代末龄幼虫取食量最大,占整个取食量的80%左右,4~5龄幼虫是防治的关键龄期。     

Differences in Feeding Associations of Benthophagous Fishes in two Locations

The feeding associations of two wrasse species (Pseudolabrus; Labridae) with Goniistius zonatus (Cheilodactylidae) in two temperate water locations (Morode and Arakashi) in southern Japan were compared. At both locations, the wrasses accompanied G. zonatus so as to eat benthic invertebrates flushed out by the latter's feeding activities. Although the average feeding frequency of G. zonatus did not differ between the two locations, the host was more frequently and persistently followed at Morode, where foraging hosts often stayed at a calcareous-algal patch for a significant time and repeatedly pecked at the same site (repeated-feeding). At Arakashi, however, characterised by a poor calcareous-algal mat, G. zonatus usually took single sporadic bites at the substrate other than the algal mat while moving about (single-feeding). At both locations, the wrasses accompanied G. zonatus at higher rates when the latter was performing 'repeated-feeding' compared with 'single-feeding', with more frequent pecking by the attendant wrasses in the former association. The differences in feeding association between the two locations apparently resulted from the different patterns of feeding behavior by the host, in turn affected by the feeding substrate composition at each location.     

Experimentally induced elevations in acid phosphatase activity in hemolymph of Biomphalaria glabrata (Mollusca).

The levels of acid phosphatase activity in the hemocytes and serum of the following four groups of the pulmonate gastropod Biomphalaria glabrata were ascertained: (1) those injected with heat-killed Bacillus megaterium, (2) those challenged with sterile distilled water, (3) those shaminjected, and (4) those left untampered. It was determined that challenge with heat-killed bacteria sesulted in significant elevations in acid phosphatase activity in both cells and serum at 1, 2, and 4 hr postinjection, with the level being highest at 2 hr. Injection with water resulted in a significant elevation in cellular enzyme level at 1 hr postinjection but not at 2 and 4 hr; however, there were significant elevations at 2 and 4 hr in serum. This is interpreted to indicate that the elevated intracellular enzyme was subsequently released into serum. Sham injection resulted in significant elevations in acid phosphatase levels in hemocytes at 2 and 4 hr postinjection but no increase of enzyme activity in serum during the course of the experiment. This is interpreted to mean that this hydrolase was not released as a result of sham injection. The source of acid phosphatase was apparently the cytoplasmic granules of granulocytes, which are true lysosomes.     

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.     

Estimation of total hemolymph volume in the horseshoe crab Limulus polyphemus

Biomedical companies extract blood from the horseshoe crab, Limulus polyphemus, for the production of Limulus Amebocyte Lysate, used worldwide for detecting endotoxins in injectable solutions and medical devices. Despite the extensive use of horseshoe crabs by the biomedical industry, total hemolymph volume for this species is not known. The hemolymph volume of 60 adult horseshoe crabs was estimated using an inulin dilution technique. Blood volume of the horseshoe crab represented as a percentage of wet body weight was 25?±?2.2% for males and 25?±?5.1% (mean?±?SD) for females. Relationships between hemolymph volume and weight (p?=?0.0026, r ²?=?0.8762), hemolymph volume and prosomal width (p?<?0.0001), and hemolymph volume and inter-ocular width (p?<?0.0001) were observed. No significant differences were observed between males and females. The relationship of animal size and hemolymph volume can be used to predict how much blood can be drawn from horseshoe crabs used by the biomedical industry, and can be of further use in future bleeding mortality studies.     

Anti-apoptotic mechanism of silkworm hemolymph in HeLa cell apoptosis

Silkworm hemolymph (SH) was found to exhibit anti-apoptotic activities in mammalian and insect cell systems. An anti-apoptotic mechanism of SH was investigated in a staurosporine-induced HeLa cell using flow cytometry, caspase assay, Immunoblot, and Immunochemistry. The addition of 5% SH to the medium resulted in lower intracellular activities of caspase-3 and caspase-9 after 0.6 μM of staurosporine treatment; however, SH did not directly inhibit the activities of those enzymes. This suggests SH inhibits the event upstream of these caspase activation steps, such as mitochondrial level events. We found from Immunoblot and Immunochemistry that cytochrome c release from the mitochondria was blocked by SH. SH also inhibited Bax translocation to the mitochondria. On the contrary, SH did not block the apoptosis when Bax is not involved in promoting apoptosis. With these results, we propose that SH protects mitochondria from apoptosis signal via blocking Bax translocation, and the subsequent apoptotic events are then inhibited. The inhibition of apoptosis using SH and its components may lead to new approaches for the minimization of cell death during commercial animal cell cultures.     

Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata

Lie K. J., Jeong K. H. and Heyneman D. 1980. Inducement of miracidia-immobilizing substance in the hemolymph of Biomphalaria glabrata. Intemational Journal for Parasitology10: 183–188. More than 85% of echinostome-infected albino B. glabrata laboratory strain snails develop miracidia-immobilizing substance(s) (MIS) in the hemolymph, while less than 5% of control uninfected snails show this ability. Snails infected with Echinostoma lindoense show a strong miracidial immobilizing test (MIT) when homologous miracidia are exposed to the hemolymph and a moderate response when E. liei and Paryphostomum segregatum miracidia are used. Infection with E. paraensei results in a high level of hemolymph MIS with E. lindoense miracidia, a moderate one with P. segregatum miracidia, and a weak one when hemolymph is tested against E. liei as well as the homologous E. paraensei miracidia. Infection with E. liei induces a strong MIT with E. lindoense miracidia whereas only a moderate one was observed when using homologous or P. segregatum miracidia. Infection with P. segregatum gives a moderate MIT reaction to miracidia of the homologous species, as well as to E. lindoense and E. liei, and only a weak response to E. paraensei miracidia. Infection with S. mansoni fails to induce hemolymph that shows MIS to any of the parasites tested. Production of hemolymph MIS is temporary. It begins one day postexposure, reaches its maximum 10–14 days postexposure, and declines to the preinfection level several weeks later. Infection of snails with irradiated parasites also results in a temporary production of hemolymph MIS.Uninfected snails show a tissue-extract MIS, which is especially strong when digestive gland extracts are used. However, these snails give little or no evidence of a hemolymph MIS.     

韩江下游底栖动物的分布及其对水质的评价

韩江下游底栖动物的分布及其对水质的评价蓝宗辉（韩山师范学院生物系,广东潮州５２１０４１）ＴｈｅＺｏｏｂｅｎｔｈｏｓＤｉｓｔｒｉｂｕｔｉｏｎｉｎｔｈｅＬｏｗｅｒＲｅａｃｈｅｓｏｆＨａｎｊｉａｎｇＲｉｖｅｒａｎｄＡｓｅｓｍｅｎｔｏｆｉｔｓＷａｔｅｒＱｕａ...     

中华甲虫蒲螨寄生双条杉天牛幼虫血淋巴变化

为了解中华甲虫蒲螨Pyemotes zhonghuajia Yu and Zhang对双条杉天牛Semanotus bifasciatus(Motschulsky)的致死过程,将已发育成熟的中华甲虫蒲螨膨腹体移入放有双条杉天牛2龄幼虫的玻璃指形管中,在蒲螨的一个生活史60h内,观察其取食行为,并在移入后每隔12h用考马斯亮蓝法分别测定天牛幼虫和蒲螨血淋巴中总蛋白质含量。结果显示,蒲螨蛋白含量总体呈现上升的趋势,在36～48h上升幅度最大,达61.78%;而天牛幼虫血淋巴总蛋白质含量则呈总体下降趋势,与对照相比,在36～48h之间下降最为明显;蒲螨总蛋白质含量在24h后处理组与对照组差异均为极显著,天牛幼虫总蛋白质含量在48h后处理组与对照组差异显著,这表明蒲螨在24h已搜寻到寄主开始取食,且取食效果明显,而天牛幼虫在48h后表现为麻痹致死,36～48h蒲螨取食对天牛致死起关键作用。12h时处理组的双条杉天牛幼虫总蛋白质含量比对照组略高,推测是由于幼虫对外寄生物蒲螨具有一定的免疫防御反应。     

Feeding behavior and related functional morphology of the mayflyEphemerella needhami (Ephemeroptera: Ephemerellidae)

The feeding behavior and functional morphology associated with feeding in Ephemerella needhamiMcDunnough larvae were studied using videomacroscopic techniques, gut content analysis, and scanning electron microscopy. Two stereotypic feeding cycles were employed by the larvae. In the maxillary brushing cycle, the maxillae are the primary food-gathering organs, with the main food being detritus deposited on the filamentous alga Cladophora.In the mandibular biting cycle, the mandibles are the primary food-gathering organs used to bite Cladophora filaments.Epiphytic diatoms on Cladophorawere another important part of the diet. Behavioral similarities are apparent in the choreography and synchronization of mouthpart movements among mayflies from several families. Functional morphological comparisons are drawn with hypognathous E. needhami, Cloeon dipterum, Baetis rhodani,and Siphlonurus aestivalis, aswell as the prognathous Heptageniidae. Differences in mouthpart usage and structure are related to the relative development of setal fields and combs and the feeding microhabitat.