Studies on very-high-density lipoprotein of Triatoma infestans hemolymph in relation to its function as free fatty acid carrier

The function of Triatoma infestans very-high-density lipoprotein (VHDL) as a free fatty acid transport protein was analyzed. Lipophorin (HDLp) and VHDL are the unique hemolymphatic proteins able to transport free fatty acids (FFA). The transfer of this lipid species between HDLp and VHDL was studied using ¹⁴C-palmitic acid-labeled VHDL or ¹⁴C-palmitic acid-labeled HDLp as donor substrate and the same unlabeled lipoproteins as acceptor substrate. The VHDL is more effective as acceptor of ¹⁴C-FFA from HDLp rather than donor of ¹⁴C-FFA to HDLp. When ¹⁴C-palmitic acid-labeled VHDL was incubated with either fat body or testicle, it was observed that the ¹⁴C-palmitic acid was taken up by both tissues and incorporated into their lipid components.     

粘虫幼虫血淋巴脂蛋白的分离鉴定及理化特性分析

通过利用NaBr密度梯度超速离心对粘虫Mythimna separata幼虫的血脂蛋白进行了分离及理化特性分析。查明了幼虫血淋巴内主要是高密度脂蛋白(HDLp),而低密度脂蛋白(LDLp)的含量极低。这两种脂蛋白的密度分别为HDlp 1.123g/Ml,LDLp 1.059g/mL。HDLp和LDLp蛋白含量、脂类、氨基酸的种类测定结果表明HDLp,LDLp的蛋白含量分别是25.2μg/μL和11μg/μg。HDLp水解后可获得16种氨基酸。LDLp可获得15种。氨基酸的种类比较一致,其含量变化幅度也不大。HDLp脂类部分占最多的是二酰基甘油酯(DC)和磷脂(PC)。其余的脂类依次是碳氢化合物(HC)和胆固醇?。LDLp主要是磷脂和碳氢化合物。昆虫脂蛋白的主要中性甘油酯是二酰基甘油酯。     

In vitro lipid transfer between lipoproteins and midgut-diverticula in the spider Polybetes pythagoricus

It has been already reported that most hemolymphatic lipids in the spider Polybetes pythagoricus are transported by HDL1 and VHDL lipoproteins. We studied in vitro the lipid transfer among midgut-diverticula (M-diverticula), and either hemolymph or purified lipoproteins as well as between hemolymphatic lipoproteins. M-diverticula and hemolymph were labeled by in vivo ¹⁴C-palmitic acid injection. In vitro incubations were performed between M-diverticula and either hemolymph or isolated lipoproteins. Hemolymph lipid uptake was associated to HDL1 (67%) and VHDL (32%). Release from hemolymph towards M-diverticula showed the opposite trend, VHDL 75% and HDL1 45%. Isolated lipoproteins showed a similar behavior to that observed with whole hemolymph. Lipid transfer between lipoproteins showed that HDL1 transfer more ¹⁴C-lipids to VHDL than vice versa. Only 38% FFA and 18% TAG were transferred from M-diverticula to lipoproteins, while on the contrary 75% and 73% of these lipids, respectively, were taken up from hemolymph. A similar trend was observed regarding lipoprotein phospholipids. This study supports the hypothesis that HDL1 and hemocyanin-containing VHDL are involved in the uptake and release of FFA, phospholipids and triacylglycerols in the spider P. pythagoricus. The data support a directional flow of lipids from HDL1 and VHDL suggesting a mode of lipid transport between lipoproteins and M-diverticula.     

Lipophorin levels in the yellow fever mosquito,Aedes aegypti,and the effect of feeding

High density lipophorin (HDLp) is the major lipid transport vehicle in insect hemolymph. Using an indirect ELISA, levels of HDLp were measured in the yellow fever mosquito, Aedes aegypti. The level of lipophorin, when normalized to the total weight of the insect, was similar in the different developmental stages. Starvation (access to water only) of adult females did not affect the level of HDLp nor its density when compared to sugar-fed females. On the other hand, blood feeding (of normally sugar-fed females) resulted in a three-fold increase of the HDLp level at 40 h after feeding. This increase was accompanied by a slight but significant increase in the density of HDLp at 24 h after feeding. Ingestion of a lipid-free protein meal or a lipid-supplemented protein meal induced changes in HDLp level and density that were comparable to those induced by ingestion of a blood meal. Ingestion of a blood meal, following starvation (access to water only) from the moment of adult emergence, did not induce an increase in HDLp level. The results presented indicate that, in contrast to other insect species, A. aegypti responds to an increased need for lipid transport in the hemolymph by increasing the amount of HDLp. Arch. Insect Biochem. Physiol. 34:301–312, 1997. © 1997 Wiley-Liss, Inc.     

Lipid transfer particle catalyzes transfer of carotenoids between lipophorins of Bombyx mori

The yellow color of Bombyx mori hemolymph is due to the presence of carotenoids, which are primarily associated with lipophorin particles. Carotenoids were extracted from high density lipophorin (HDLp) of B. mori and analyzed by HPLC. HDLp contained 33 μg of carotenoids per mg protein. Over 90% of carotenoids were lutein while -carotene and β-carotene were minor components. When larval hemolymph was subjected to density gradient ultracentrifugation, a second minor yellow band was present, which was identified as B. mori lipid transfer particle (LTP). During other life stages examined however, this second band was not visible. To determine if coloration of LTP may fluctuate during development, we determined its concentration in hemolymph and compared it to that of lipophorin. Both proteins were present during all life stages and their concentrations gradually increased. The ratio of lipophorin: LTP was 1015:1 during the fourth and fifth instar larval stages, and 2030:1 during the pupal and adult stages. Thus, there was no correlation between the yellow color attributed to LTP and its hemolymph concentration. It is possible that yellow coloration of the LTP fraction corresponds to developmental stages when the particle is active in carotene transport. To determine if LTP is capable of facilitating carotene transfer, we took advantage of a white hemolymph B. mori strain which, when fed artificial diet containing a low carotene content, gives rise to a lipophorin that is nearly colorless. A spectrophotometric, carotene specific, transfer assay was developed which employed wild type, carotene-rich HDLp as donor particle and colorless low density lipophorin, derived from the white hemolymph strain animals, as acceptor particle. In incubations lacking LTP carotenes remained associated with HDLp while inclusion of LTP induced a redistribution of carotenes between the donor and acceptor in a time and concentration dependent manner. Time course studies suggested the rate of LTP-mediated carotene transfer was relatively slow, requiring up to 4 h to reach equilibrium. By contrast, studies employing ³H-diacylglycerol labeled HDLp as donor particle in lipid transfer assays revealed a rapid equilibration of label between the particles. Thus, it is plausible that the slower rate of LTP-mediated carotene transfer is due to its probable sequestration in the core of HDLp.     

Purification and properties of a storage protein from the hemolymph of Rhodnius prolixus

A non-arylphorin hexameric storage protein (SPR) has been isolated from the larval hemolymph of the bug, Rhodnius prolixus. The purified protein contained 0.69% lipid and 1.04% carbohydrate. SPR had a mol. wt of 470,000, with a subunit mol. wt of 78,000. In electron micrographs, SPR molecules appeared very similar to calliphorin. After the metamorphic molt, SPR persisted at lower concentrations in the hemolymph of adult insects, and it disappeared only in fasting bugs.     

Wax moth,Galleria mellonella fat body receptor for high-density lipophorin (HDLp)

To identify and characterize the HDLp (high-density lipophorin) receptor from Galleria mellonella (LpRGm), we used techniques of ligand blotting. This method was, to our knowledge, first used to characterize the lipophorin receptor (LpR) in insects. LpRGm had an approximate molecular weight of 97 kDa under non-reducing conditions and bound the HDLp specifically. The time-course of lipophorin binding to their receptor protein was rapid. The binding of lipophorins to their receptors was saturable with a Kd of 34.33+/-4.67 microg/ml. Although Ca2+ was essentially required in the binding of HDLp to their receptors, interestingly increasing concentration of Ca2+ has shown to have a slight inhibitory effect. EDTA was used here as Ca2+ chelating reagent, because Mg2+ in the binding buffer did not affect the binding of HDLp to their receptors, and inhibited the binding of HDLp and LpRGm absolutely. Suramin (polysulfated polycyclic hydrocarbon), known to inhibit the binding of lipoproteins to their receptors, effectively abolished the binding of HDLp to their receptors. LpRGm showed the stage specific binding activity especially in day 1-3 last instar larval, prepupal, and day 1-3 adult stages.     

The Conjugated Plasma Proteins of the American Cockroach : II. Changes during the molting and clotting processes

Evidence has been presented for the possible transport function of conjugated roach plasma proteins during molting. Extensive changes in these proteins are evident during the premolt stage. The remainder of the instar appears to be devoted to a gradual return to the intermolt stage. This recovery process is characterized principally by a transformation of less mobile lipoproteins to lipoproteins of higher electrophoretic mobilities and may be indicative of a lipoprotein-lipase reaction in insects. This transformation also appears to give rise to a second sex-specific lipoprotein. Significant changes in the glycoproteins at the premolt and ecdysial stages may indicate transport of carbohydrate for storage or utilization. A total of six fractions has been shown to occur in roach plasma in the period from one molt to the next. The process of clotting appeared to be concerned primarily with the blood lipoproteins. These protein fractions reacted to form two new lipoproteins, one of which was the relatively insoluble coagulum network. The second major protein fraction appearing as a result of the coagulation process also was a lipoprotein which contained a lower concentration of carbohydrate than the clot. A possible method of assaying or screening new anticoagulants for insect blood is proposed.     

Juvenile hormone-binding protein and juvenile hormone esterase activity in hemolymph from last-instar nymphs of Leucophaea maderae

The concentration of the juvenile hormone-binding protein (JHB) in hemolymph was determined throughout the last nymphal instar. It was found to be 3.9 μM at the molt to the instar, rising to 13 μM by mid-instar, and dropping to 6.7μM the day before emergence. Endocrine control of its production during the last nymphal instar could not be established. The apparent juvenile hormone esterase (JHF) activity was low at the molt to the last instar, but rose about fivefold by mid-instar, and then modestly declined. On the day of emergence, JHF activity rose to the highest level observed. A four- to fivefold increase in absolute JHF activity was determined during the first half of the last nymphal instar. This increase is not regulated by JH. Removal of the JHB from hemolymph samples by precipitation with a polyclonal specific antibody increased the JHF activity up to 1,000-fold. Thus, changes in the concentrations of JHB can affect the apparent activity of JHE, which is unrelated to the production or degradation of the JHF.     

Quantitative changes and synthesis of cyanoprotein in whole body and tissues during development of the bean bug,Riptortus clavatus

Changes in biliverdin-binding cyanoprotein content in whole body and tissue extracts during development of nymphal and adult (non-diapause) bean bugs, Riptortus clavatus were analyzed by rocket immunoelectrophoresis (RIE). RIE using anti-CPegg serum can be used to determine the content of CP-A (Cp-1, 2 and 3) and CP-B (CP-4) separately. During the nymphal stage CP content of whole body changes cyclically in each instar. In the first nymphal instar, CPegg is the main CP which disappears during the first-second instar ecdysis. In nymphal bugs from the 2nd to 4th instars only CP-B (CP-4) is detected, and at the beginning of each instar the CP content is very low but increases toward the next ecdysis, after which CP decreases and disappears very rapidly. In the 5th nymphal instar, CP-B is the major CP but CP-A (CP-1, 2 and 3) is also detected. These changes in whole body CP content of 5th instar nymphs are observed in both females and males. The content of total CPs in the 5th instar nymph reaches about 1000 μg in the whole insect. During nymphal-adult ecdysis, nymphal CPs decrease and disappear at day 2 after emergence. In female adults CP-A (CP-1 only) increases rapidly after day 4 of adult emergence, while no CP is detected in male adults. In females CPs were detected only in the fat body, hemolymph and ovary. In the mid-5th-instar nymphs, CPs (CP-A and B) are mainly distributed in the hemolymph. CPs in the Hemolymph decrease during nymphal-adult ecdysis, whereas they increase in the fat body. CPs disappear from both the hemolymph and fat body by 2 days after ecdysis. Subsequently in the adult stage only CP-A increases again in the fat body and ovary. By tracer experiments using [³⁵S]-methionine, the fat body was shown to be the site of CP synthesis. CP-A and B synthetic activity was detected in nymphal females whereas, only CP-A synthesis was observed in adult females, while no CP synthesis was seen in adult males.     

New Functions of Arthropod Bursicon: Inducing Deposition and Thickening of New Cuticle and Hemocyte Granulation in the Blue Crab,Callinectes sapidus

Arthropod growth requires molt-associated changes in softness and stiffness of the cuticle that protects from desiccation, infection and injury. Cuticle hardening in insects depends on the blood-borne hormone, bursicon (Burs), although it has never been determined in hemolymph. Whilst also having Burs, decapod crustaceans reiterate molting many more times during their longer life span and are encased in a calcified exoskeleton, which after molting undergoes similar initial cuticle hardening processes as in insects. We investigated the role of homologous crustacean Burs in cuticular changes and growth in the blue crab, Callinectes sapidus. We found dramatic increases in size and number of Burs cells during development in paired thoracic ganglion complex (TGC) neurons with pericardial organs (POs) as neurohemal release sites. A skewed expression of Burs β/Burs α mRNA in TGC corresponds to protein contents of identified Burs β homodimer and Burs heterodimer in POs. In hemolymph, Burs is consistently present at ∼21 pM throughout the molt cycle, showing a peak of ∼89 pM at ecdysis. Since initial cuticle hardness determines the degree of molt-associated somatic increment (MSI), we applied recombinant Burs in vitro to cuticle explants of late premolt or early ecdysis. Burs stimulates cuticle thickening and granulation of hemocytes. These findings demonstrate novel cuticle-associated functions of Burs during molting, while the unambiguous and constant presence of Burs in cells and hemolymph throughout the molt cycle and life stages may implicate further functions of its homo- and heterodimer hormone isoforms in immunoprotective defense systems of arthropods.     

Enzymatic and Ultrastructural Changes in Thoracic Muscles of Triatomine Insects during the Last Stages of Metamorphosis

Dipetalogaster maximus and Triatoma infestans are hematophagous insects, vectors of Chagas' disease. After the last molt of their metamorphosis, from fifth instar nymph to adult, they acquire wings and the ability to fly, which is important for their dispersal. Some biochemical changes accompanying this last stage have been studied by determining activity of hexokinase (EC 2.7.1.1), fructose-6-phosphate kinase (EC 2.7.1.11), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glutamate dehydrogenase (EC 1.4.1.4), aspartate aminotransferase (EC 2.6.1.1), malate dehydrogenase (EC 1.1.1.37) and glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) in thoracic muscle extracts of fifth instar nymphs and adults. Activity of all the enzymes, expressed in U per mg protein, was significantly higher in muscles of adults than of nymphs, except that of aspartate aminotransferase, had lower activity in adults of T. infestans. The increase of glycerol-3-phosphate dehydrogenase activity was particularly striking (30-fold), while the increase in glucose-6-phosphate dehydrogenase activity was of a lesser magnitude than those observed for other enzymes. Comparative ultrastructural studies of thoracic muscles showed that in adult preparations mitochondria were more numerous and larger in size, and presented more cristae than in muscles of fifth instar nymphs. The biochemical changes detected appear to be the expression of the adaptation of adult muscles for flight activity. Thus, adult muscles would have higher glycolytic and respiratory capacity than those of fifth instar nymphs. The operation of systems transferring hydrogen into mitochondria, especially that of the glycerophosphate shuttle, may be greatly increased in adult muscles.     

Lipophorin of female Blattella germanica (L.): characterization and relation to hemolymph titers of juvenile hormone and hydrocarbons

High density lipophorin (HDLp) from the hemolymph of the German cockroach, Blattella germanica (L.) (Family Blattellidae), has an apparent molecular weight of 670kDa, with an isoelectric point of 7.0 and a density of 1.109g/ml. It is composed of two subunits, apolipoprotein-I (212kDa) and apolipoprotein-II (80kDa), and consists of 51.4% lipid, 46.2% protein and 2.4% carbohydrate. Hydrocarbons constitute 42.2% of the total lipids which also contain diacylglycerol, cholesterol and phospholipid. Lipophorin is rich in the amino acids glutamic acid, aspartic acid, lysine, valine, and leucine. Specificity of a polyclonal antibody was demonstrated by Western blotting and Ouchterlony immunodiffusion: the antiserum recognized native HDLp and apolipoprotein-I, but not apolipoprotein-II, purified vitellin, or other hemolymph proteins. It also recognized a protein in the hemolymph of Supella longipalpa (Blattellidae) but did not cross-react with hemolymph proteins from Periplaneta americana (Blattidae) or Diploptera punctata (Blaberidae). An enzyme-linked immunosorbent assay was developed to measure the HDLp titer in the hemolymph of adult females. The titer of HDLp, a juvenile hormone binding protein, exhibited no clear relationship to the changing titer of juvenile hormone in hemolymph. The hemolymph titer of hydrocarbon, which is also carried by HDLp, showed some functional relation to the concentration of HDLp in the hemolymph. Because it concurrently serves multiple functions in insect development and reproduction, lipophorin titer might covary with the titers of lipid ligands that occur at high concentrations and require extensive shuttling through the hemolymph.     

Cyanoprotein: Immunological properties and content changes during the development of non-diapause female bean bugs,Riptortus clavatus

Immunological properties and content changes of cyanoprotein (CP) were investigated in the bean bug, Riptortus clavatus. Anti-CPegg serum was prepared by immunizing a rabbit with CP purified from eggs (CPegg). In the Ouchterlony double diffusion test, the precipitin line between CPegg and anti-CPegg serum fused with that of non-diapause and diapause female hemolymph and anti-CPegg serum. Rocket immunoelectrophoresis (RIE) using anti-CP serum showed two types of rockets (A and B) depending on the samples. Namely, CPegg and non-diapause female adult hemolymph formed A rockets (heavy-stained with Coomassie Brilliant Blue) and early diapause female adult hemolymph formed B rockets (light-stained), but hemolymph from fifth instar nymphs formed both A and B rockets. Both rockets A and B were demonstrated by SDS-PAGE analysis of the precipitin lines to be formed from the same CP subunit (MW, 76,000). CP-1, 2 and 3 bands from native PAGE of nymphal hemolymph formed A rockets and CP-4 formed B rockets. The contents of CP-A (CP-1 to 3) and CP-B (CP-4) were separately determined by measuring the sizes of rocket A and B. CP-A and CP-B content were demonstrated to increase during the development of the last instar nymph and decrease at adult emergence by RIE analysis of non-diapause female whole body extracts. CP-B is predominant in the nymphal stage. In the early adult stage (day 2 and 3 after emergence), neither CP-A nor CP-B were detected. Only CP-A appeared again at day 4 after emergence and increased during development and vitellogenesis of non-diapause females.     

Numerical allometric growth of the ommatidia,antennal sensilla,and teeth of foretibial combs in the milkweed bug Oncopeltus fasciatus Dallas (Heteroptera : Lygaeidae)

Oncopeltus fasciatus (Heteroptera : Lygaeidae), the milkweed bug, was bred in captivity. Sampling showed that individuals grow exponentially through their 6 developmental stages with an average linear increase per molt of 42% for the females and 41% for the males. The number of ommatidia per eye grows with negative allometry from an average of 30 in the first instar to 860 in the male and 820 in the female adult. The total number of sensilla on the 2 flagellar segments of an antenna increases with negative allometry during the 5 nymphal stages from a mean of 239 in the first instar to 2462 in the last. At this point, this allometric growth pattern is sharply broken by distinct numerical increase to 7163 on the adult flagellum. The number of teeth composing the foretibial comb, the tool for grooming the distal flagellar segment, grows with negative allometry through all 6 developmental stages. Calculations using previously published data from the migratory locust, Locusta migratoria, revealed the same growth pattern of antennal sensilla: uniform allometric growth during the nymphal development, broken by a conspicuous upward jump to the adult number of sensilla. In the American cockroach, Periplaneta americana, this growth pattern of antennal sensilla holds only for the male; the female continues the nymphal allometric growth into the adult stage. These observations on allometric growth fit three theoretical explanations: 1. Smooth allometric growth is evidence for an aut-adaptation to increasing size. 2. Ex-adaptations to novel ecological niches cause breaks in allometric growth patterns. 3. Chapman's rule, which states that increased mobility correlates with greater olfactory sensitivity, correctly predicts the observed breaks in the allometric growth patterns in the abundance of antennal sensilla.     

Alternative lipid mobilization: The insect shuttle system

Lipid mobilization in long-distance flying insects has revealed a novel concept for lipid transport in the circulatory system during exercise. Similar to energy generation for sustained locomotion in mammals, the work accomplished by non-stop flight activity is powered by oxidation of free fatty acids (FFA) derived from endogenous reserves of triacylglycerol. The transport form of the lipid, however, is diacylglycerol (DAG), which is delivered to the flight muscles associated with lipoproteins. In the insect system, the multifunctional lipoprotein, high-density lipophorin (HDLp) is loaded with DAG while additionally, multiple copies of the exchangeable apolipoprotein, apoLp-III, associate with the expanding particle. As a result, lipid-enriched low-density lipophorin (LDLp) is formed. At the flight muscles, LDLp-carried DAG is hydrolyzed and FFA are imported into the muscle cells for energy generation. The depletion of DAG from LDLp results in the recovery of both HDLp and apoLp-III, which are reutilized for another cycle of DAG transport. A receptor for HDLp, identified as a novel member of the vertebrate low-density lipoprotein (LDL) receptor family, does not seem to be involved in the lipophorin shuttle mechanism operative during flight activity. In addition, endocytosis of HDLp mediated by the insect receptor does not seem to follow the classical mammalian LDL pathway.Many structural elements of the lipid mobilization system in insects are similar to those in mammals. Domain structures of apoLp-I and apoLp-II, the non-exchangeable apolipoprotein components of HDLp, are related to apoB100. ApoLp-III is a bundle of five amphipathic -helices that binds to a lipid surface very similar to the four-helix bundle of the N-terminal domain of human apoE. Despite these similarities, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different, since the TAG-rich mammalian lipoproteins play no role as a carrier of mobilized lipids during exercise and besides, these lipoproteins are not functioning as a reusable shuttle for lipid transport. On the other hand, the deviant behavior of similar molecules in a different biological system may provide a useful alternative model for studying the molecular basis of processes related to human disorders and disease.     

Alate differentiation and compound-eye development in the dry-wood termite <Emphasis Type="Italic">Neotermes koshunensis</Emphasis> (Isoptera,Kalotermitidae)

In social insects, caste-specific characters develop in the postembryonic differentiation processes. However, the mechanisms of caste-specific organ development have yet to be elucidated. In order to obtain insights into the relationship between caste differentiation and the regulation of organ development, we determined the caste-developmental pathway and observed compound-eye development accompanying alate differentiation in the dry-wood termite, Neotermes koshunensis. As previously reported in other Neotermes, this species has a linear caste-developmental pathway, comprising six larval- and two nymphal-instar stages. Although the apparent eye formation occurs during the last nymphal stages, just prior to the imaginal molt, individuals possess eye primordia from the first larval-instar stage. The outer morphological structure of the eye was observed from the third larval-instar stage. The detailed differentiation of cells constituting ommatidia appeared to occur in relatively young larval instars (fourth stage), although the pigmentation of pigment cells and detailed structural formation of ommatidia occurred during the final stage of alate development, i.e., during the late second nymphal-instar stage. This suggests that eye development is arrested in the larval stages, and then resumed during the late nymphal stage to complete functional eye formation, which is required for nuptial flight. In comparison to major hemimetabolous insects, which possess functional compound eyes even at the first instar larva, this termite species shows the heterochronic shift in terms of compound-eye development. Received 20 March 2006; revised 24 September 2006; accepted 4 October 2006.     

Immunoanalysis of unique protein in Trichoplusia ni larvae parasitized by the braconid wasp Chelonus near curvimaculatus

Parasitization in insects brings about profound biochemical and physiological effects in the host which may include complete overriding of the normal endocrinological program, resulting in precocious metamorphosis and in blockage of pupal development. The subtle effects of parasitization include changes in the expression of hemolymph proteins and the appearance of proteins which are unique to parasitized hosts. One such protein has been identified in the hemolymph of Trichoplusia ni larvae parasitized by the braconid wasp Chelonus near curvimaculatus. In this study, purified preparations of the parasitism-specific protein were used to generate polyconal antibodies against the protein. Results from the immunocharacterization indicate the antibodies obtained are highly specific for the protein and are present in a high titer (1:8000 antiserum dilution yielded strong signals in analysis of the protein in 0.25 μl hemolymph). Subsequently, the expression of the parasitism-specific protein in the hemolymph and tissues was analyzed by immunoblotting during the entire course of development in normal and parasitized insects. The parasitism-specific protein was not detected in normal, unparasitized larvae. In parasitized insects, expression of the parasitism-specific protein appears to be stage-specific in that it is only detected during the last larval stadium of precociously metamorphosing larvae, but is absent from all earlier stages of development.     

Changes in the Amounts of the Molt-Inhibiting Hormone in Sinus Glands during the Molt Cycle of the American Crayfish, Procambarus clarkii

The purposes of this study are to determine the molt cycle of the American crayfish, Procambarus clarkii, and to quantify the amounts of the molt-inhibiting hormone (Prc-MIH) in the hemolymph and neurohemal sinus glands during the molt cycle of the American crayfish. The molt cycle was classified into six stages based on the changes in volumes of gastroliths in the stomach and ecdysteroid titers in the hemolymph. A sandwich-type enzyme immunoassay using specific antibodies raised against N-terminal and C-terminal segments of Prc-MIH was developed for the Prc-MIH assay. It is sensitive to as little as 0.5 fmol of Prc-MIH (3.3 x10(-12) M). In the hemolymph, no Prc-MIH could be detected at any of the molt stages tested. However, in the sinus gland, it was demonstrated that the amount of Prc-MIH changes in a molt-stage-specific manner during the molt cycle. It was particularly noteworthy that the initiation of a molting sequence (i.e., entering the early premolt stage) corresponded to the increase in Prc-MIH content in the sinus gland, because the finding is consistent with the hypothesis that crustaceans enter the premolt stage when the MIH secretion from the sinus gland is reduced or ceases.