Oral immunization with Escherichia coli K-12 of the fifth instar larvae of the silkworm, Bombyx mori, reared on an artificial diet under completely aseptic conditions.

1. Effect of oral administration of live or formalin-treated Escherichia coli (E. coli) K-12 to the fifth instar, days 1 and 3 larvae of the silkworm, Bombyx mori, on induction of antibacterial activity in the haemolymph was investigated using the silkworms reared on an artificial diet under completely aseptic conditions. 2. When live E. coli was administered to the male day 1 larvae, low but significant antibacterial activity of 3.8 mm was detectable in the haemolymph of one individual at 48 hr after immunization. The proportion of the larvae to express antibacterial activity increased thereafter and at 120 hr after immunization, all three individuals showed antibacterial activity. In day 3 male larvae, activity was detectable at 48 and 72 hr after immunization. 3. When formalin-treated E. coli was orally administered to days 1 and 3 male larvae, no activity was detectable at any time post-immunization. 4. In the second experiment, when day 1 larvae, females and males were orally immunized with live E. coli, only females showed antibacterial activity in the haemolymph, beginning from 24 hr after immunization and up to 96 hr. 5. Removal of an antibiotic, chloramphenicol, from ingredients of an artificial diet was required for induction of antibacterial activity with oral administration of live E. coli. 6. When live E. coli that grows at pH 9.0 was selected and used for oral immunization, antibacterial activity was induced both in females and males at 72 hr after immunization and the activity was observed at 96 hr. 7. These results suggest that establishment of oral immunization with live E. coli in the silkworm larvae requires multiplication of E. coli in the midgut lumen and possibly its colonization on the luminal surface.     

Determination of Caffeic Acid in the Digestive Juice of Silkworm Larvae and Its Antibacterial Activity against the Pathogenic Streptococcus faecalis AD–4

Caffeic acid (CA) was identified as an antibacterial substance in the digestive juice of silkworm larvae. Concentrations of CA in the digestive juice decreased from 460 ppm to 3 ppm as the starvation period of the larvae was prolonged from 1 hr to 12 hr. The specimens of digestive juice (ca. pH 10.0) containing 460 ppm and 222 ppm of CA had antibacterial activity against Streptococcus faecalis AD–4, a pathogenic intestinal bacterium affecting silkworm larvae, and an alkaline medium (pH 10.0) containing 500 ppm of authentic CA also exhibited antibacterial activity.

These findings suggest that CA plays an important role in the defense mechanism of silkworm larvae against the bacterial infection.     

Hormonal control of uric acid storage in the fat body during last-larval instar of Manduca sexta

In the last-larval instar of the tobacco hornworm, Manduca sexta, a switch from excretion of uric acid to storage in the fat body occurs during transition from the feeding to the wandering stage. Neuroendocrine control of this change from excretion to storage was demonstrated by neck-ligation experiments with synchronously reared larvae. Results indicate that a neurohormone is released from the head 24–30 hr before the initiation of wandering and coincident with the first release of ecdysone that initiates metamorphosis. Direct involvement of the moulting hormone was shown by the effects of multiple injections of 20-hydroxyecdysone into the abdomen of larvae that had been ligated before the release of hormone. Urate levels in the fat body were 20- to 100-fold higher from hormone-injected larvae as from saline inject controls. Topically applied juvenile hormone or methoprene reversed the 20-hydroxyecdysone-induced storage of urate. Increased levels of uric acid in the haemolymph during pupal development result from the presence of juvenile hormone, and the abrupt decrease in uric acid concentration in the haemolymph just prior to pupal ecdysis results from a decreased titre of juvenile hormone. Applications of methoprene prevented the decrease in uric acid levels in the haemolymph.     

家蚕感染蛹虫草后的生理生化变化

蛹虫草分生孢子侵染5龄家蚕Bombyx mori后,家蚕血淋巴中总糖、海藻糖、蛋白质和甘油酯含量均有不同程度的下降,其中甘油酯含量下降最为明显。海藻糖酶活性在侵染初期也明显降低。接种后家蚕体内的保护酶超氧化物歧化酶、过氧化物酶和过氧化氢酶活性也有较大变化,其中超氧化物歧化酶活性上升最为明显,在4日内由441.841 U/mL升至601.255 U/mL。     

Comparison of parasitism by Cotesia glomerata with bacterial infection and wounding in Pieris brassicae: induction of new haemolymph polypeptides and changes in humoral immune response

In Pieris brassicae, parasitism by Cotesia glomerata and bacterial infection are differentiated with respect to haemolymph protein arrays, and production or suppression of antibacterial agents. Bacteriolytic activity in haemolymph from parasitized larvae was slightly, but significantly, higher 24h post-treatment than that of untreated and wounded controls. Micrococcus lysodeikticus- or lipopolysaccharide-(LPS) injected insects exhibited an 11-fold greater response than those parasitized. At 24h post-treatment, antibacterial activity against Escherichia coli was observed in haemolymph from all but untreated larvae. Injection of Grace's medium, M. lysodeikticus or LPS, caused a greater than threefold response than parasitization or wounding. The protein banding patterns of parasitized hosts did not correspond to those of the other treatments. Two parasitoid-induced proteins (38 and 128 kDa) were examined. Both were found in parasitized insects, not in those wounded, injected with Grace's medium, M. lysodeikticus or LPS. Neither protein was bacteriolytic or bacteriostatic in inhibition zone assays.     

荧光定量PCR检测家蚕核型多角体病毒在其宿主体内的增殖动态

为了研究家蚕核型多角体病毒(Bombyx mori nuclear polyhedrosis virus,BmNPV)在其宿主幼虫体内不同组织中的增殖动态,对敏感性家蚕品种306幼虫进行经口定量滴注病毒。在接种后9个时间点,对中肠、血淋巴和脂肪体进行取样。以BmNPV DNA 聚合酶基因(dnapol)指示病毒拷贝数,同时以家蚕细胞质肌动蛋白A3(actin A3)基因作为参比基因,用荧光定量PCR的方法分别检测各个时间点的中肠、血淋巴和脂肪体中病毒的拷贝数。结果表明经口感染2 h,病毒进入中肠;12 h,病毒已经到达血淋巴和脂肪体;再经过约12 h的潜伏期,病毒在各组织中开始快速增殖,到84 h各组织中病毒增殖达到平台期。     

人白细胞介素-11基因在家蚕培养细胞和虫体内的表达

将缺少编码信号肽序列的人白细胞介素－11（hIL-11)546核苷酸cDNA,重组于质粒pBacPAK8构建重组转移载体pBacIL-11,与经线性化修饰的家蚕核型多角体病毒（BmBacPAK)DNA共转染家蚕培养细胞株BmN,获得了插入hIL-11基因的重组病毒。Southern杂交表明重组病毒基因组中含有hIL-11基因片段,RNA斑点杂交表明hIL-11基因得到了转录。重组病毒感BmN细胞株、家蚕幼虫和蛹,在细胞培养上清、细胞抽提物、幼虫和蛹的体液样品中,SDS－PAGE电泳分析都能检测得到表达产物的特异性条带;采用IL－11依赖细胞株B9－11和MTT法测定表达产物的生物活性,表明rIL-11基因分别在培养细胞和蚕体内得到了高效表达。     

中蜂抗菌物质的诱导

本文报道了用大肠杆菌注射处理中蜂Ａｐｉｓ ｃｅｒａｎａ Ｆａｂｒｉｃｉｕｓ后,用含菌培养基平板测活法,就中蜂抗菌物质的产生规律及抗菌活性进行了初步研究。结果表明,不同诱导源均可诱导中蜂产生抗菌物质,但诱导的抗菌物质的抗菌活性则有一定的差异。用大肠杆菌重复诱导则使中蜂的成活率和抗菌物质的抗菌活性有很大提高。注射大肠杆菌后冲蜂抗菌物持产生高峰在４８小时左右。诱导后产生的抗菌物质具有广谱性,对大肠杆菌Ａｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ、枯草芽孢杆菌 Ｂａｃｉｌｌｕｓ　 ｓｕｂｔｉｌｉｓ、苏云金杆菌 Ｂａｃｉｌｌｕｓ　ｔｈｕｒｉｎｇ－ｉｅｎｓｉｓ、巨大芽孢杆菌 Ｂａｃｉｌｌｕｓ　ｍｅｇａｔｈｅｒｉｕｍ、黑腐菌 Ｘａｎｔｈｏｍｏｎａｓ ｃａｍｐｅｓｔｒｉｓ　等均有抑菌作用,而对真菌白僵菌Ｂｅａｕｖｅｒｉａ　ｂａｓｓｉａｎａ未发现抑菌作用。中蜂麻醉方法以冷冻法最为简便、易行。     

Studies on the role of protein kinase A in humoral immune response of Galleria mellonella larvae

Protein kinase A (PKA) activity was detected in the fat body of Galleria mellonella larvae by a non-radioactive method using a specific peptide substrate-kemptide. The enzyme activity was stimulated by cAMP and its analogues: BzcMP, 8-Chl-cAMP and 8-Br-cAMP in concentrations of 1-4muM. Cyclic GMP was not effective in PKA activation. A two-fold increase in PKA activity was detected in the fat body of G. mellonella LPS-challenged larvae. Selective, membrane-permeable PKA inhibitors, H89 and Rp-8-Br-cAMPS, inhibited protein kinase A activity in the fat body of G. mellonella larvae in vitro and in vivo. The inhibition of PKA activity in vivo was correlated with a considerable lowering of haemolymph antibacterial activity and a decrease in lysozyme content in the fat body of immune challenged larvae. The use of phospho-motif antibodies recognising PKA phosphorylation consensus site allowed identification of four potential PKA phosphorylation substrates of 79, 45, 40 and 36kDa in G. mellonella fat body.     

Uptake and fate of specific antibody in feeding larvae of the sheep blowfly,Lucilia cuprina

Abstract. The quantity of specific antibody ingested by larvae of Lucilia cuprina and its fate after ingestion were studied in larvae grown on sheep and on an artificial diet. Larvae grown to late first or early second instar on sheep vaccinated with horse myoglobin contained 66% less specific antibody detected by enzyme linked immunosorbent assay than larvae grown to a similar stage on an artificial diet containing 75% serum from the same sheep. A similar result was obtained when larvae were grown to mid-third instar. Larvae grown on sheep to first or second instar contained approximately the same quantity of specific antibody per unit weight of larvae as those grown to third instar. Larvae grown on diet to third instar contained 22% less specific antibody per unit weight than those grown to first or second instar. In larvae grown on diet to late third instar, ingested diet retained 91 ± 12% of its original specific antibody activity in the crop, 50 ± 11% in the anterior midgut, 8 ± 2% in the posterior midgut and 13 ± 6% in the hindgut. The mean concentration of total immunoglobulin detectable in the haemolymph of individual third instar larvae grown on diet was 1.7 ± 2.8 ug/ml. Assays of specific antibody in the haemolymph of similarly reared larvae indicated that all or most of this immunoglobulin remained functional. The implications of the quantities and distribution of ingested functional antibody found in feeding larvae of L.cuprina are discussed in relation to the possibility of vaccinating sheep against these larvae and the selection of likely internal targets as sources of potential protective antigens.     

家蚕中的5-氧脯氨酸酶及保幼激素类似物对它的影响

本工作首次在家蚕Bombyx mori的马氏管、中肠、丝腺及脂肪体等组织中测到了γ-谷氨酰循环中一个关键酶——5-L-氧脯氨酸酶的活力.该酶以马氏管中活力最高,在蚕的中肠、血淋巴中均存在有游离的5-氧脯氨酸.观察了保幼激素类似物(JHA)处理后,家蚕中肠、丝腺和脂肪体中5-L-氧脯氨酸酶活力的变化,同时观察了血淋巴中5-氧脯氨酸含量的变化.对该酶及γ-谷氨酰循环在蚕体中氨基酸转运上的可能作用进行了讨论.     

The anal portion as a salt-excreting organ in a seawater mosquito larva,A?des togoi Theobald

Summary Larvae of the marine mosquito,Aëdes togoi Theobald, tolerate environmental salinities ranging from fresh water to 300% sea water. When they were transferred from fresh water to sea water, sodium concentration in the haemolymph increased for the first 2 days and decreased to the seawater-adapted level within 4 days. When transferred from sea water to fresh water, the sodium concentration decreased markedly for the first 2 days and attained the freshwater adapted level after 4 days. When the larvae in sea water were ligated near the anus, they died within 3 days, showing an increased sodium level in the haemolymph. The larvae ligated at the neck lost considerable body weight and died within 4 days.When the anal portion, a terminal portion of the hindgut, was catheterized, the larvae maintained in sea water showed an increase in haemolymph sodium. The anal portion epithelium of the larvae adapted to 100 and 150% sea water demonstrated a strong positive reaction to the histochemical assay for chloride ions, whereas the reaction was negative or weakly positive in freshwater adapted larvae. In the larvae with the anal papillae ligated, a slight increase in haemolymph sodium occurred while in sea water. The anal papillae were weakly positive to chloride ions. Unlike salt-water mosquito larvae of the other species, in which the rectum is considered to be involved in hyperosmotic urine production and the anal papillae appear to be the extrarenal organ, the anal portion inA. togoi larvae seems to play an important role in excretion of excess ions when placed in hyperosmotic media.     

The biochemical effects of potassium chloride on the silkworm, （ Bombyx mori L.）

The supplementation with 50, 100 and 150μg/mL potassium chloride to the fifth instar larvae of the silkworm Bombyx mori on fat body glycogen, protein, total lipids and haemolymph protein and trehalose were analyzed. The fat body glycogen and protein and haemolymph protein were increased significantly in all the treated groups; whereas fat body total lipids increased only in 100 and 150μg/mL and haemolymph trehalose increased only in 150μg/mL potassium chloride-treated groups when compared with those of the corresponding parameters of the carrier controls.     

Different rate of protein granule formation in the larval fat body of Drosophila melanogaster

The concentration of protein granules was determined cytologically in different regions of the fat body during the latter half of the third larval instar of Drosophila melanogaster. The measurements made at 6 hr intervals from 72–96 hr larvae showed that the concentration of the granules was the highest in the posterior, lowest in the anterior and intermediate in the middle region of the fat body. From these measurements, it was shown that the rate of granule formation was different in each region. Furthermore, there is a strong indication that at any given stage, the rate increases gradually and continuously from the anterior to the posterior region. When the fat body from larvae prior to the time of granule formation was cultured for three days in ecdysterone-containing medium, protein granules were produced in the anterior, middle and posterior regions in the same concentration as that in 90 hr larvae. The same gradient of protein granule formation in vitro is found whether the fat body is cultured as an intact piece or as three separate, dissected regions. The putative adaptive advantage of region-dependent granule formation is discussed.     

The physiological basis of anterior inhibition of puparium formation in ligated fly larvae

Fly larvae ligated after the critical period of ecdysone release frequently fail to pupariate in the anterior part. Evidence is presented against anterior inhibition being caused by a hormonal imbalance, such as a decreased titre of ecdysone, an increased titre of juvenile hormone, and/or a lack of the neurohormonal factors that accelerate the process of pupariation. It is unlikely that the anterior inhibition results from a sustained stimulus from the sense organs, or from the presence of an inhibitory substance. The oxygen consumption in the inhibited anterior parts is 25 per cent of that in the anterior parts that pupariate. This suggests that the inhibition of pupariation is a result of oxygen deficiency. When anteriorly inhibited larvae are subjected to pure oxygen the inhibited anterior ends that would never tan in air commence to tan. The dissection of anteriorly inhibited larvae showed that the tracheal trunks were severed at the point of ligation and that the tracheae were filled with haemolymph. In the pupariated posterior parts the tracheae showed melanotic plugs at the severed ends of the tracheae preventing the flooding. Reducing the injury, by careful ligation, or by immobilizing the anterior ends by cold or tetrodotoxin reduces the incidence of anterior inhibition. Thus, anterior inhibition results from a lack of oxygen caused by the injury to the tracheal system during ligation.     

Haemolymphal activation of protyrosinase and the site of synthesis of haemolymph protyrosinase in larvae of the fleshfly, Sarcophaga barbata

When whole blood from 5 day third instar larvae of the fleshfly, Sarcophaga barbata was incubated under nitrogen at 25°C for 16 hr in the presence of salivary glands there was an increase in its protyrosinase content, which amounted to 53% of that which occurs in vivo over the same period. The protyrosinase in ammonium sulphate fractions of haemolymph that were allowed to stand at 4°C for 24 hr following the incubation at 25°C was found to have autoactivated. Analysis of all these fractions revealed the presence of a protyrosinase activator in the 30% saturated ammonium sulphate fraction. When proenzyme and haemolymph activator were mixed there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme, but was independent of the activator concentration and was comparable to that obtained using the cuticle activator. The level of activator in the haemolymph increased as larvae aged from 4 to 7 days.The effect of several compounds on the catecholase activity of the activated haemolymph protyrosinase and on the cuticle enzyme is reported and the significance of haemolymphal activation of protyrosinase is discussed.     

不同家蚕品系的耐热性特征

研究了家蚕Bombyx mori 3个品系(Pure Mysore,NB4D2和CSR2）5龄幼虫和蛹在不同温度（35,38和40℃）下的耐热性,采用Probit分析测定了它们在各温度下的LT50值和置信限。结果表明：多化性品系Pure Mysore在高温下的存活率高于两个二化性品系NB4D2和CSR2,而两个二化性品系中NB4D2表现出更好的耐热性。家蚕幼虫接触38℃高温6 h和40℃高温3 h后,其血淋巴中出现90,70和29 kDa的热激蛋白条带。在恢复过程中,NB4D2和CSR2的血淋巴中未见29 kDa蛋白条带,而Pure Mysore幼虫的血淋巴中29 kDa蛋白仍然表达。当幼虫置于高温下时,血淋巴中90和70 kDa蛋白表达,但是检测不到29 kDa蛋白。研究认为热激蛋白表达与热带家蚕不同品系的耐热性以及与同一品系不同发育阶段的耐热性具有相关性。     

RH-5992--an ecdysone agonist on model system of the silkworm Bombyx mori

RH-5992 is a novel synthetic non-steroidal ecdysteroid agonist with a high selectivity towards Lepidopteran species. The effect of ecdysone agonist RH-5992 on larval period, larval weight, silk gland weight and haemolymph protein profile were examined in the model organism, the larvae of silkworm, Bombyx mori. The LD50 values were found to be 16.21 and 12.01 micrograms/larva for 72 and 96 hr respectively. In the present study, three sublethal concentrations of 1/5th, 1/10th and 1/20th of LD50 at 72 hr were selected and applied on the mid-dorsal line of the silkworm B. mori. The maximum mortality of 35% was observed in the group which received the highest (3.2 micrograms/larva) concentration of RH-5992. The mortality rate was found to be dose dependent as well as time dependent. Interesting results were observed in haemolymph profile of the RH-5992 treated larvae as staining intensity of 30 kDa protein decreased significantly whereas the effect was not marked on other major proteins like storage proteins and vitellogenin polypeptides. From the results, it is confirmed that RH-5992 causes changes in larval characters and protein profile of silkworm B. mori. It is proposed that RH-5992 may cause negative effect specifically on reproductive characters like development of ovary and egg production due to decrease in 30 kDa protein.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

饲料中维生素和无机盐对rBmNPV-Bm表达系统植酸酶基因表达活性的影响

【目的】为查明宿主的营养对重组家蚕杆状病毒-家蚕表达系统(rBmNPV-Bm)外源基因表达活性的影响,探寻提高家蚕Bombyx mori生物反应器产率的新途径。【方法】以家蚕BmNPV病毒为表达载体,以人工饲料饲养的5龄家蚕为宿主,以植酸酶基因为报告基因,探讨了家蚕人工饲料中维生素和无机盐对外源基因表达产物活性的影响。【结果】家蚕血淋巴中外源植酸酶的活性随着饲料中维生素和无机盐添加量的不同而发生显著变化。在本试验设区范围内,当维生素C的含量为饲料干物质量的1 %,B族维生素混合物为0.25 %,无机盐混合物为1 %,维生素B6为100 g饲料干物中含2 mg时,血淋巴中植酸酶活性达到最大值。【结论】通过改变家蚕饲料中微量营养成分的含量是提高rBmNPV-Bm表达系统外源基因表达活性的有效途径。