Binding of juvenile hormone III to lipophorin from the american cockroach Periplaneta americana

Whole hemolymph from the American cockroach, Periplaneta americana, efficiently binds juvenile hormone (JH) III and to a lesser extent JH-I and 10, 11-epoxyfarnesyl diazoacetate (EFDA). The dissociation constants for racemic JH-III and EFDA are 30 ± 2 nM and 1.0 μM, respectively. Isolated lipophorin also binds [³H]JH-III and to a lesser extent JH-I. Other proteins from the hemolymph do not bind JH-III. Binding of JH-III to lipophorin is enantioselective. The dissociation constant, measured with a 92% 10R and 8% 10S mixture, is 21 ± 2 nM. Each lipophorin molecule contains one specific binding site for JH-III. It is concluded that lipophorin is the JH-III-specific transport protein in the hemolymph of the American cockroach. By a combination of photoaffinity labelling and gradient electrophoresis with sodium dodecyl sulphate on polyacrylamide gel, we showed that the JH-III-specific binding site is probably located on apolipophorin I.     

Sugar binding properties of the vitellogenic proteins in the Colorado beetle,demonstrated by hemagglutination and precipitation experiments

Summary The sugar binding properties of 2 important vitellogenic proteins in Colorado beetle hemolymph were demonstrated by hemagglutination and precipitation experiments. The agglutination of human red blood cells by the hemolymph of reproducing females was observed up to a hemolymph dilution of 1/256, irrespective of the blood-group. It increased significantly after trypsinization of the crythrocytes. Vitellogenin 1 was identified as the hemagglutinin. Hemagglutination and hemagglutination inhibition tests showed that this protein has a low affinity for hexosamines and a higher affinity for sulfated polysaccharides. Precipitation tests demonstrated that besides vitellogenin, another major yolk protein, chromoprotein 2, reacts with sulfated polysaccharides. The possibility that there is a specific reaction of the vitellogenic proteins with well defined saccharides on the oocyte surface is discussed. This lectintype reaction may explain the selectivity of yolk precursor endocytosis.     

Molecular characteristics of lipophorin,the juvenile hormone-binding protein in the hemolymph of the Colorado potato beetle

Lipophorin, the protein that specifically binds juvenile hormone in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, is a high-density lipoprotein of M_r ～ 574,000. Lipophorin contains 43% lipid and is composed of two apoproteins: apolipophorin I (M_r ～ 251,000) and apolipophorin II (M_r ～ 78,000). Both apoproteins contain mannose residues. Carotenoids make up a substantial part of the lipid fraction. Lipophorin constitutes about 25% of the total hemolymph proteins. Its concentration in the hemolymph (26 μM in 4-day-old long-day and 40 μM in 4-day-old short-day beetles) changes with different physiological conditions concomitant with changes in total protein content. Lipophorin specifically binds 10R-juvenile hormone III with high affinity. The dissociation constant for 10R-juvenile hormone III is 12 ± 2 nM. One lipophorin molecule contains one specific juvenile hormone-binding site. The concentration of binding sites therefore equals that of lipophorin in hemolymph.     

Development and partial characterization of monoclonal antibodies to the hemolymph juvenile hormone binding protein of Manduca sexta

Murine monoclonal antibodies were made against the hemolymph juvenile hormone binding protein (JHBP) of Manduca sexta. Binding studies in conjunction with Western blot analysis of native and sodium dodecyl sulfate gels confirmed that antibodies from 10 hybridoma lines interacted with the juvenile hormone binding protein. The pattern of cross-reactivity among the hybridoma lines suggests that different epitopes are recognized. The cross-reactivity pattern for monoclonal antibody 9 suggested a common epitope in three different hemolymph proteins: JHBP, insecticyanin and a 40–45 kDa protein. Western blot analysis of a two-dimensional gel using monoclonal antibody 6 revealed interaction with JHBP and with several proteins that may be precursors or degradation products of the binding protein. An enzyme-immunoassay was developed that detects JHBP in the hemolymph at nanogram levels.     

High-molecular-weight serum proteins from Locusta migratoria: identification of a protein specifically binding juvenile hormone III

ABSTRACT. Tritiated 10,11-epoxyfarnesyl diazoacetate (EFDA), a photoaffinity label, can be covalently attached to the binding site of a JH-III-specific binding protein in the haemolymph of Locusta migratoria migratorioides (R & F). The specificity of the binding of EFDA to the binding protein is verified by displacement with excess unlabelled JH-III, and EFDA can be used to identify the binding protein in native pore-limiting gradient poly(acrylamide) gel electrophoresis (PAGE) and sodium dodecyl sulphate-PAGE. The native binding protein has a molecular weight of 575,000 and is composed of seemingly identical subunits of molecular weight 81,000.
Three other high-molecular weight serum proteins are identified by native PAGE: a lipophorin, composed of two kinds of apolipophorins, a larval storage protein and a cyanoprotein. The molecular weights and subunit structures of these proteins are investigated, but none of these other high-molecular weight proteins bind JH-III to an appreciable extent.     

An Insect Multiligand Recognition Protein Functions as an Opsonin for the Phagocytosis of Microorganisms

We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and β-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.     

Juvenile hormone binding protein traffic — Interaction with ATP synthase and lipid transfer proteins

Juvenile hormone (JH) controls insect development, metamorphosis and reproduction. In insect hemolymph a significant proportion of JH is bound to juvenile hormone binding protein (JHBP), which serves as a carrier supplying the hormone to the target tissues. To shed some light on JHBP passage within insect tissues, the interaction of this carrier with other proteins from Galleria mellonella (Lepidoptera) was investigated. Our studies revealed the presence of JHBP within the tracheal epithelium and fat body cells in both the membrane and cytoplasmic sections. We found that the interaction between JHBP and membrane proteins occurs with saturation kinetics and is specific and reversible. ATP synthase was indicated as a JHBP membrane binding protein based upon SPR-BIA and MS analysis. It was found that in G. mellonella fat body, this enzyme is present in mitochondrial fraction, plasma membranes and cytosol as well. In the model system containing bovine F₁ ATP synthase and JHBP, the interaction between these two components occurs with K_d = 0.86 nM. In hemolymph we detected JHBP binding to apolipophorin, arylphorin and hexamerin. These results provide the first demonstration of the physical interaction of JHBP with membrane and hemolymph proteins which can be involved in JHBP molecule traffic.     

GP-9s Are Ubiquitous Proteins Unlikely Involved in Olfactory Mediation of Social Organization in the Red Imported Fire Ant,Solenopsis invicta

The red imported fire ant (RIFA), Solenopsis invicta, is an invasive species, accidentally introduced in the United States that can cause painful (sometimes life-threatening) stings to human, pets, and livestock. Their colonies have two social forms: monogyne and polygyne that have a single and multiple functional queens, respectively. A major gene (Gp-9), identified as a putative pheromone-binding protein on the basis of a modest amino acid sequence identity, has been suggested to influence the expression of colony social organization. Monogyne queens are reported to possess only the GP-9B alleles, whereas polygyne queens possess both GP-9B and GP-9b. Thus, both social forms are reported to express GP-9B, with GP-9b being a marker expressed in polygynes but it is absent in monogynes. Here, we report two types of polygyne colonies, one that does not express GP-9b (monogyne-like) and the other expressing both proteins, GP-9B and GP-9b. Given their expression pattern, GP-9s are hemolymph proteins, which are more likely to be involved in the transport of lipids and small ligands within the homocoel. GP-9B existed in two forms, one of them is phosphorylated. The helical-rich content of the protein resembles the secondary structures of a beetle hemolymph protein and moth pheromone-binding proteins. An olfactory role is unlikely given the lack of specific expression in the sensillar lymph. In marked contrast to GP-9s, a chemosensory protein, SinvCSP, is demonstrated to be specifically expressed in the antennae. Within the antennae, expression of SinvCSP is restricted to the last two segments, which are known to house olfactory sensilla.     

A new method of detecting hormone-binding proteins electroblotted onto glass fiber filter: juvenile hormone-binding proteins from grasshopper hemolymph

We have developed a new method to identify juvenile hormone (JH)-binding proteins blotted onto glass fiber filter (GFF) after electrophoretic separation. Insect JH regulates reproduction in the two-striped grasshopper, Melanoplus bivittatus. A number of proteins are involved in the delivery of JH from its site of synthesis to the nuclei of fat body cells where it acts to induce vitellogenesis. To identify JH binding proteins, hemolymph was separated by PAGE, electroblotted onto GFF, and incubated in [10-3H]JH-III. The amount of hormone bound by blotted proteins increased with the amount of protein on the filter, was competitively displaced by excess non-labeled hormone, and was affiliated with individual bands on fluorograms of proteins blotted after electrophoretic separation. GFF etched with trifluoroacetic acid was better than nitrocellulose, Zeta Probe, cellulose acetate or unetched GFF. Phosphate (pH 6.0-7.3) or Tris buffers (pH 7.3-8.0) worked equally well for the procedure. Unbound hormone was easily removed by short washes in buffer, and adequate binding for detection was achieved in a 15 min incubation. Preliminary data suggest that this technique may be used to detect receptors, carriers, and binding proteins of steroid hormones.     

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.     

Storage proteins are present in the hemolymph from larvae and adults of the Colorado potato beetle.

The protein composition of larval and adult hemolymph from the Colorado potato beetle, Leptinotarsa decemlineata, was investigated and some abundant, high molecular weight proteins were identified and characterized. Diapause protein 1, which occurs in the hemolymph of last instar larvae and short-day adults, appeared to be a storage protein. This protein dissociated into two bands due to the high pH used in nondenaturing gels. Its quaternary structure was established by chemical crosslinking. It appeared to be a hexamer. Diapause protein 1 is composed of approximately 82,000 subunits. The amino acid composition and N-terminal sequence of this protein has been determined. Specific antibodies against diapause protein 1 have been developed. Topical application of 1 microgram pyriproxyfen, a juvenile hormone analog, to last instar larvae and short-day adults suppressed the appearance of this protein in the hemolymph. Pyriproxyfen prematurely induced vitellogenin, when applied to last instar larvae. A larval specific protein was also identified in the hemolymph. Its temporary appearance in the hemolymph of last instar larvae, its subunit composition (M(r) approximately 82,000) and its suppression by pyriproxyfen suggests that this protein is a storage protein as well.     

Synthesis and secretion of a precursor hemolymph juvenile hormone-binding protein in the adult female cockroach Leucophaea maderae

Hemolymph juvenile hormone-binding protein (JHBP) is synthesized and secreted from fat body in the adult female cockroach, Leucophaea maderae. The data in this paper suggest it is initially secreted from the fat body as a larger peptide whereas data in the accompanying paper demonstrate that JHBP is apolipophorin I. Using media from cultures of fat body maintained in vitro, a JH-binding component was found that is JH III saturable, has a K_D of 1.5 × 10⁻⁸ M, binds JH III > JH II > JH I, and has a sedimentation value of 6.5S on high salt sucrose gradients. Each of these properties is identical to those of the JHBP extracted from the hemolymph. To identify the protein that bound JH, media proteins were photoaffinity labeled with 10-[10,11-³H]epoxyfarnesyl diazoacetate ([³H]EFDA). The results revealed that two media proteins bound [³H]EFDA in the absence of JH III, but not in the presence of 100-fold excess JH III. The molecular weights of the two media peptides were estimated by SDS-PAGE to be 275,000 and 220,000.To determine if the JHBP found in media of fat body cultures was due to hemolymph contamination of fat body, incorporation of [³H]leucine into newly synthesized and secreted fat body proteins during a 48 h culture period was monitored. During the culture period, linear increases in the concentrations of radiolabeled 275 and 220 kD JHBP were observed. Monoclonal antibodies specific for the 220 kD hemolymph JHBP were found to recognize both the 275 and 220 kD JHBPs in the media.To investigate the possibility that the 275 kD protein is a precursor to the 220 kD protein and that components of the hemolymph process or modify the precursor, hemolymph was introduced into fat body cultures and relative concentrations of the 275 and 220 kD media JHBPs were determined. Addition of hemolymph to these organ cultures resulted in an increase in the concentration of radiolabeled 220 kD JHBP and a proportional decrease in the concentration of radiolabeled 275 kD JHBP, suggesting that the 275 kD protein is a precursor to the 220 kD hemolymph JHBP. The mechanism of processing or modification remains undetermined.     

Characterization of juvenile hormone-binding proteins of hemolymph of Locusta migratoria

The binding of juvenile hormone (JH) by components from hemolymph of adult female Locusta migratoria was characterized to establish whether hemolymph JH-binding proteins could be distinguished from a protein of fat body (BP-1) that may be a JH receptor. Hemolymph was analyzed by the hydroxyapatite assay, gel separation chromatography, polyacrylamide gel electrophoresis, and density gradient centrifugation. Three fractions that bound JH were separated from whole hemolymph by DEAE cellulose column chromatography, and these differed from all three cytosol-binding components. The major hemolymph component (H-A) showed relatively stable binding of JH, a slight loss of binding capacity after delipidation, and a K_d for JH-I of 16 nM. The K_ds for JH-l and JH-lll with unfractionated hemolymph were 26 and 42 nM respectively. The order of effectiveness of competitors for binding of [³H]JH-l was JH-lll > JH-l ? methoprene > hydroprene ? acids of methoprene and hydroprene. The data indicated that unlabeled JH-lll was bound more effectively than its radioactive counterpart. The sedimentation values determined by sucrose density gradient ultracentrifugation were 13-14 S for hemolymph, and the sedimentation value was not altered by the inclusion of 0.4 M KCl throughout the gradient. The data indicated that H-A resembled the specific JH carriers and differed from the putative receptor of fat body cytosol by several criteria.     

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.     

BBE31 from the Lyme disease agent Borrelia burgdorferi,known to play an important role in successful colonization of the mammalian host,shows the ability to bind glutathione

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi sensu lato complex spirochetes. The spirochete is located in the gut of the tick; as the infected tick starts the blood meal, the spirochete must travel through the hemolymph to the salivary glands, where it can spread to and infect the new host organism. In this study, we determined the crystal structures of the key outer surface protein BBE31 from B. burgdorferi and its orthologous protein BSE31 (BSPA14S_RS05060 gene product) from B. spielmanii. BBE31 is known to be important for the transfer of B. burgdorferi from the gut to the hemolymph in the tick after a tick bite. While BBE31 exerts its function by interacting with the Ixodes scapularis tick gut protein TRE31, structural and mass spectrometry data revealed that BBE31 has a glutathione (GSH) covalently attached to Cys142 suggesting that the protein may have acquired some additional functions in contrast to its orthologous protein BSE31, which lacks any interactions with GSH. In the current study, in addition to analyzing the potential reasons for GSH binding, the three-dimensional structure of BBE31 provides new insights into the molecular details of the transmission process as the protein plays an important role in the initial phase before the spirochete is physically transferred to the new host. This knowledge will be potentially used for the development of new strategies to fight against Lyme disease.     

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。     

The role of endogenous antifreeze protein enhancers in the hemolymph thermal hysteresis activity of the beetle Dendroides canadensis

Antifreeze proteins (AFPs) lower the freezing point of water by a non-colligative mechanism, but do not lower the melting point, therefore producing a difference between the freezing and melting points termed thermal hysteresis. Thermal hysteresis activity (THA) of AFPs from overwintering larvae of the beetle Dendroides canadensis is dependent upon AFP concentration and the presence of enhancers of THA which may be either other proteins or low molecular mass enhancers. The purpose of this study was to determine the relative contributions of endogenous enhancers in winter D. canadensis hemolymph.Winter hemolymph collected over four successive winters (1997-1998 to 2000-2001) was tested. The first three of these winters were the warmest on record in this area, while December of the final year was the coldest on record. Protein and low molecular mass enhancers raised hemolymph THA 60-97% and 35-55%, respectively, based on hemolymph with peak THA for each year collected over the four successive winters. However, the hemolymph AFPs were not maximally enhanced since addition of the potent enhancer citrate (at non-physiologically high levels) resulted in large increases in THA. ¹³NMR showed that glycerol was the only low molecular mass solute present in sufficiently high concentrations in the hemolymph to function as an enhancer. Maximum THA appears to be ∼8.5 °C.     

Changes in the hemolymph lipophorin and very high density lipoprotein levels during the fifth nymphal and adult stages of Triatoma infestans

The occurrence of lipophorin (HDLp) and a very high density lipoprotein (VHDL) throughout the last nymphal and adult stages of male T. infestans was followed by quantitative immunodiffusion using specific antisera derived from the purified lipoproteins of adult male insects. Significant changes on the concentration of these hemolymph lipoproteins (Lps) occurred during the mentioned developmental stages. These changes include the following. A great accumulation of hemolymph proteins and Lps, particularly VHDL, during the nymphal stage. This increase reached a peak just before the last molt, where the concentration of VHDL and HDLp were 74 and 42 mg/ml hemolymph, respectively. A sharp decline in the Lps concentration just after the molt and during the first 2 weeks of adult life. These changes imply a six-fold decrease on the VHDL and a two-fold decrease in the HDLp concentrations. Another increase in the protein concentration that begins around the third week of the adult stage affecting both Lps, but mainly the HDLp. From its hexameric structure, amino acid composition and high concentration in nymphal stages, the VHDL of T. infestans could be considered a storage protein. The fact that VHDL persists in a considerable concentration in the hemolymph of adult insects differenciates this VHDL from this protein. The distribution of ¹⁴C free fatty acid (FFA) among the hemolymph proteins of T. infestans shows that the FFA are associated only with VHDL and HDLp. The developmental changes of the Lps pattern are accompanied by changes in the relative distribution of FFA between these Lps. The VHDL is the principal carrier of FFA during the fifth-nymph stage while HDLp is the main protein and FFA-carrier in adult life.     

Hemolymph proteins in ticks

In comparison to insects and Crustacea, our knowledge of the predominant hemolymph proteins in ticks is minimal. The hemolymph protein most studied in ticks has been vitellogenin (Vg). Vg is synthesized by the tick fat body after female adults obtain a blood meal, is released into the hemolymph and is absorbed by developing oocytes as vitellin (Vn). Much of what we know about Vg is from studies of Vn. In general, the carbohydrate, lipid and amino acid composition is similar to insects except that in the tick, Vg contains heme, most likely from the digestion of host hemoglobin. In the American dog tick, Dermacentor variabilis, Vg is comprised of two native proteins and seven subunits on SDS-PAGE. Vg has been characterized in five tick species but the amino acid sequence is not yet available. Another predominant hemolymph protein, apparently a carrier protein (CP), has recently been studied in two tick species. This protein is found in the hemolymph of both male and females adults, in adult tissues outside of the hemolymph in some tick species, in coxal fluid of soft ticks and in whole body homogenates from eggs, larvae and nymphs. CP from the hard tick, D. variabilis, contains cholesterol, phospholipids, monoacylglycerides, triacylglycerides, free fatty acids, carbohydrate and heme. Under identical assay conditions, the analogous protein in the soft tick, Ornithodoros parkeri, did not contain heme. CP in the American dog tick consists of two subunits, one of which has 61% identity to the biliprotein, artemocyanin, from the fairy shrimp. CP is identical to a heme-lipoprotein (HeLp) from Boophilus microplus. The exact roles of CP and HeLp have not yet been fully determined, but they apparently are important in heme sequestration and as a storage depot for protein and lipid. Macroglobulin, lectin, antimicrobial, JH binding, JH esterase, and other tick hemolymph proteins are also discussed.     

Activité comparée des hormones juvéniles en C-18 (JH-I) et en C-16 (JH-III) chez Locusta migratoria

The comparative activity of C-16 and C-18 juvenile hormones is studied in Locusta migratoria on four well-known physiological functions of the corpora allata by means of a single injection of a solution of hormone in oil at doses of 50, 100, and 200 μg/animal. Judged on morphogenesis and pigmentation, JH-I (C-18 JH) as well as JH-III (C-16 JH) show a real juvenilizing effect. The potency of JH-I is much higher than that of JH-III because the first hormone only produces supernumerary larvae and most modified green animals. JH-I counterbalances exactly the lack of CA on the gonadotropic function whereas JH-III allows only about 50 per cent development of oöcytes. The cardiotropic activity of JH-I is similar to that of the CA. The C-18 juvenile hormone is until now the only studied ‘juvenilizing’ compound which increases the heartbeat. JH-III appears to have no noticeable effect on the heart.These results combine to prove that only JH-I has an activity similar to the Locusta corpora allata on morphogenesis, pigmentation, ovarian maturation, and the cardiac activity of L. migratoria.