The synthesis and biological activity of linear and cyclic analogs of the two diuretic peptides of Diploptera punctata

We have investigated the effect of analogs of the two Dippu diuretic hormones, Dippu-DH(46) and Dippu-DH(31), on fluid secretion by Malpighian tubules of male Diploptera punctata. We synthesized analogs containing the amino acid methyl-homoserine, to replace methionine residues, to render these modified peptides less subject to oxidation. We have also synthesized C-terminal fragments and their corresponding cyclic analogs to determine their effect on fluid secretion in D. punctata. Our results indicate that the modified peptides retain significant activity in the Ramsay secretion assay. The linear fragments displayed no activity or some inhibitory activity whereas the cyclic analog fragments showed stimulatory activity, in the case of DH(46), or slight inhibitory activity, in the case of DH(31).     

Beetle diuretic peptides: the response of mealworm (Tenebrio molitor) Malpighian tubules to synthetic peptides, and cross-reactivity studies with a dung beetle (Onthophagus gazella)

This paper reports the effects of different diuretic factors on the Malpighian tubules of beetles. Calcitonin (CT)-like peptides from silkmoth and mosquito increase fluid secretion in a dose-dependent manner in the tubules of Tenebrio molitor, but the cockroach CT-like peptide, Dippu-DH(31), has no effect. Thapsigargin induces a small but significant increase in tubule secretion rates. The interactions between different factors in mealworm tubules were explored by testing CT-like peptides, thapsigargin and the mealworm CRF-related diuretic factor Tenmo-DH(37) in various combinations, but no synergistic effects were observed. C-terminal fragments of the CRF-related diuretic peptides Locmi-DH(46) and Dippu-DH(46) fail to increase fluid secretion in mealworm tubules, unlike their corresponding whole peptides. Cross-reactivity of factors between beetle species was investigated using the scarabaeid Onthophagus gazella. Tenmo-DH(37) increases fluid secretion in isolated tubules of O. gazella in a dose-dependent manner, revealing a high degree of cross-reactivity in this distantly related beetle species. However, homogenates of O. gazella brains inhibited fluid secretion in mealworm tubules.     

Presence and activity of a Dippu-DH31-like peptide in the blood-feeding bug, Rhodnius prolixus

The blood-feeding bug, Rhodnius prolixus, ingests large blood meals, then undergoes a period of rapid diuresis which is under neurohormonal control. In both cockroach (Diploptera punctata) and fruit fly (Drosophila melanogaster) a calcitonin-like DH31 neuropeptide has been identified [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74] and demonstrated to be active on Malpighian tubule secretion [Coast GM, Webster SG, Schegg KM, Tobe SS, Schooley DA. The Drosophila melanogaster homologue of an insect calcitonin-like diuretic peptide stimulates V-ATPase activity in fruit fly Malpighian tubules. J Exp Biol 2001;204:1795-804; Furuya K, Milchak RJ, Schegg KM, Zhang J, Tobe SS, Coast GM, et al. Cockroach diuretic hormones: characterization of a calcitonin-like peptide in insects. Proc Natl Acad Sci USA 2000;97:6469-74]. Using an antibody raised against D. punctata (Dippu) DH31, we demonstrate the presence of Dippu-DH31-like immunoreactivity in the CNS, salivary glands, hindgut and neurohemal sites of 5th instar Rhodnius. Double-label immunohistochemistry for Dippu-DH31-like and serotonin-like immunoreactivity demonstrates some co-localization of these factors in cells of the mesothoracic ganglionic mass (MTGM) and in neurohemal sites on the abdominal nerves. When tested on Rhodnius 5th instar Malpighian tubules, Dippu-DH31 stimulated minor increases in rate of secretion. Dippu-DH31 tested in combination with serotonin resulted in increases in the rate of secretion which were at least additive.     

A study of signal transduction for the two diuretic peptides of Diploptera punctata

We investigated second messengers involved in the action of the CRF-related peptide Dippu-DH46 and the calcitonin-like peptide Dippu-DH31 in Diploptera punctata. Dippu-DH46 causes a dose-dependent increase in intracellular cAMP levels, its diuretic activity is mimicked by cAMP agonists, but is attenuated by Rp-cAMPS. Dippu-DH46 acts synergistically with kinins and thapsigargin; both mobilize intracellular Ca2+. Dippu-DH46 also acts synergistically with cAMP agonists, and its effect is inhibited by a PKC inhibitor, suggesting it also activates intracellular Ca2+. Dippu-DH31 has no effect on cAMP levels and its activity is not blocked by cAMP agonists. Neither peptide stimulated cGMP levels in a dose-dependent manner, nor does cGMP have any effect on fluid secretion.     

The synthesis of an analogue of the locust CRF-like diuretic peptide, and the biological activities of this and some C-terminal fragments

The synthesis is described of an analogue of the locust CRF-like diuretic peptide in which methionine in positions 1,3, and 13 is replaced by isosteric methyl-homoserine residues. This analogue has been tested for biological activity on Malpighian tubules in vitro, and feeding behavior in vivo. It is highly active in stimulating fluid secretion and accumulation of cAMP in tubules, and on increasing the latency to feed and reducing meal duration. A 15 residue fragment from the C-terminus of the CRF-like peptide, Locmi-DP(32-46), is fully active in the feeding assay, but has only weak ability to stimulate the accumulation of cAMP in tubules. Two smaller fragments, Locmi-DP(32-37) and Locmi-DP(41-46), were tested but neither had consistent biological activity in any of the assays used here. None of the peptides tested have any substantive activity in increasing cGMP in tubules.     

Locusts as model organisms in which to study immunogen-induced anorectic behaviour

When injected into adult or nymphal Locusta that have been deprived of food for 2 h, immunogens such as laminarin and bacterial LPS can induce an almost immediate dose-dependent state of anorexia for at least 1 h. Such anorexia is a component of a medley of physiological and behavioural changes called collectively ‘sickness behaviour’ that occurs in a wide range of animals in response to infection or immune challenge. Sub-optimal amounts of injected laminarin allow some locusts to feed, but with a longer latency than in controls, although the length of the first meal is unaffected. The feeding behaviour of fifth instar nymphs is more sensitive to laminarin than that of adults, but both stages respond to amounts of immunogen that are lower than those required to activate the phenoloxidase cascade. Injection of adipokinetic hormone (AKH) before the period of food deprivation prevents the anorexigenic action of the laminarin in adults but not in nymphs. It is argued that the effect of the AKH may be indirect, through its lipid-mobilising action. The insecticide pymetrozine increases the latency to feed but also reduces the length of the first meal, and its anorexigenic activity is not affected by injection of AKH. The present data support the concept that laminarin-induced anorexia involves a central lack of motivation to eat, rather than a ‘stop eating’ signal. Others have shown that the mechanism of action of pymetrozine involves the serotonergic system and can be blocked by mianserin, so it is intriguing that in the present study injection of mianserin prior to that of laminarin modulates the anorexigenic effect of the immunogen. This suggests that biogenic amines are involved in the control of appetitive behaviour in locusts, as they are in vertebrates. The possible usefulness of the locust model in studying sickness-induced anorexia is discussed briefly.     

Pathogenicity of six isolates of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina, Hyphomycetes) to Perillus bioculatus (F) (Hem., Pentatomidae)

Abstract:  The two-spotted stink bug, Perillus bioculatus (F) (Hem., Pentatomidae) is considered an important predator of Colorado potato beetle egg masses and small larvae. The susceptibility of P. bioculatus second instar nymphs to six isolates of Beauveria bassiana (Balsamo) Vuillemin, which are highly pathogenic to Colorado potato beetle, was tested in the laboratory. Five isolates were highly pathogenic and caused more than 77% mortality after 8 days at a concentration of 10⁶ conidia/ml. However, isolate IPP46 showed low pathogenicity and caused only 11% mortality of nymphs. The pathogenicity of isolates was independent of their host, source or country of origin. Isolate IPP46 was tested at six different concentrations. A linear relationship between the concentration of B. bassiana and the mortality of nymphs was observed. The 50% lethal concentration (LC₅₀) value was 3.4 × 10⁷ conidia/ml. The LT₅₀ decreased with increase of conidia concentration used. The present results suggest that isolate IPP46 has good potential as a biological control agent within an integrated pest management programme.     

Tentacle contraction in Heliophrya erhardi (suctoria): The role of inositol phospholipid metabolites and cyclic nucleotides in stimulus-response coupling

Extracellular stimulation of Heliophrya erhardi with 15 V, 100 ms induces tentacle contraction. The effects of a number of pharmacological agents on response latency (time from stimulus to start of contraction) and contraction time (time for tentacles to reach 20% of their original length) were recorded. Contraction was enhanced in the presence of phorbol ester TPA and R59022, both of which increase activation of diacylglycerol-dependent protein kinase C. Lithium ions, which are known to inhibit dephosphorylation of inositol 1-phosphate, also affected the response, causing a decrease in latency and increase in contraction time. These results strongly implicate products of inositol phospholipid metabolism in stimulus-contraction coupling. The latency of contraction was reduced in the presence of IBMX, a nonspecific inhibitor of cyclic nucleotide phosphodiesterases. Application of membrane-permeable cyclic nucleotide analogs led to an increase in latency and contraction time for cAMP analog, but a decrease in both parameters for cGMP. This suggests that the two cyclic nucleotides have opposing roles in modulating the response. The possible roles and interactions of membrane phospholipid metabolites and cyclic nucleotides in the control of tentacle contraction of H. erhardi are discussed.     

Caste differences in feeding and trophallaxis in the western drywood termite, Incisitermes minor (Hagen) (Isoptera, Kalotermitidae)

Summary: Differences in feeding and trophallaxis among castes of the western drywood termite, Incisitermes minor (Hagen), were determined using rubidium (Rb) as a tracer. Both 5th- and 6th-instar nymphs and 3rd- and 4th-instar larvae fed directly on Rb-treated paper and acted as both donors and recipients in trophallactic exchanges with other larvae and nymphs, and as donors for soldiers and alates. Soldiers and alates did not feed on Rb-treated paper suggesting that they do not feed directly on wood and are completely dependent on nymphs for their nutritional needs. Larvae paired with Rb-fed nymphs received the greatest amount of Rb suggesting that there may be a hierarchy of trophallaxis in drywood termites. Transfer efficiency, the percentage of the total Rb intake of donors that is passed to the recipients, ranged from 1.1 % (nymphal donors to alate recipients) to 16.6 % (larval donors to nymphal recipients).     

Effect of water temperature,feeding frequency,and protein percent in the diet on water quality,growth and behavior of Nile tilapia Oreochromis niloticus (Linnaeus, 1758)

This study evaluated how water temperature (26, 28, and 30°C), number of meals per day (one or two meals), and protein percent in diet (20, 25 and 30%) impact growth performance, biometric indices, and feeding behavior of Nile tilapia, Oreochromis niloticus. Fish were randomly allocated into 18 equal replicate groups. Higher final body weight was observed in fish reared at 30°C and fed one meal per day containing 30% crude protein. Better weight gain, weight gain %, feed conversion ratio, specific growth rate, and condition factor were recorded in fish reared at 26°C and fed one meal per day containing 30% protein. The best length weight relationship was obtained in fish reared at 26°C and fed one meal per day containing 30% crude protein. Shorter feeding duration and duration of appetite inhibition latency were recorded in fish reared at 30°C, fed one meal per day, and given a diet containing 30% protein. The highest proactivity was recorded in fish reared at 30°C, received one meal per day, and with 25% crude protein in their diet. Conclusively, rearing Nile tilapia at 26–30°C with a lower feeding frequency (one meal/day) and a 30% crude protein diet achieved better performance and feeding behavior.     

Synthesis and characterization of cyclic and linear helical poly(alpha-peptoids)s by N-heterocyclic carbene-mediated ring-opening polymerizations of N-substituted N-carboxyanhydrides

Cyclic poly(alpha-peptoid)s [a.k.a. poly(N-R-glycine)] with chiral aromatic side-chains [R = (R)- or (S)-CHMePh] have been synthesized by N-heterocyclic carbene-mediated ring-opening polymerization of N-substituted N-carboxyanhydrides (N(R-NCA)). Their linear analogs have been prepared by primary amine-initiated polymerization of the corresponding N(R-NCA). Poly[(R)/(S)-N-CHMePh-glycine] with polymer molecular weights (MWs) in the range of 4-15 kg mol(-1) and low MW distribution (Polydispersity index (PDI) < 1.15) can be readily accessed by these methods. Their high MW analogs were not obtained due to the competitive formation of cyclic oligomeric species that result from intramolecular transamidation. Intrinsic viscosity measurements confirm the architectural difference between the polymers prepared by the two methods and reveals that both cyclic and linear poly[(S)-N-CHMePh-glycine]s behave as random-coil polymers in 0.1M LiBr/Dimethylformamide (DMF) solution. Circular dichroism analysis suggests that the cyclic and linear poly(alpha-peptoid)s retain polyproline I helix conformations, analogously to previously reported linear oligomers. Differential scanning calorimetry analysis reveals that cyclic and linear poly[(S)-N-CHMePh-glycine] are both amorphous with the glass transition temperature of the cyclic polymers (T(g) = 122 degrees C) being notably higher than that of the linear analogs (T(g) = 112 degrees C) with identical MW. These results are consistent with the absence of chain ends, causing the polymers to have reduced segmental mobilities.     

Dietary items of predacious aquatic bugs (Nepoidea: Heteroptera) in Japanese wetlands

Some species of predatory aquatic bugs (Nepoidea, including Belostomatidae and Nepidae) are designated as threatened-vulnerable species in the Red Data Book of Japan and are regarded as effective predators of freshwater snails and mosquito larvae. To reveal the feeding habits of Nepoidea, we investigated their dietary items in wetlands and obtained data from the published literature. Lethocerus deyrolli (Vuillefroy) preys mainly upon frogs, and the major diet of the nymphs is tadpoles. Laccotrephes japonensis (Scott) adults prey mainly upon tadpoles. Lethocerus deyrolli nymphs and L. japonensis adults compete against each other as same-guild members because they are sympatric in wetlands. Both nymphs and adults of Appasus japonicus (Vuillefroy) feed on snails, and L. japonensis nymphs feed on aquatic insects, including mosquito larvae (39.3% of aquatic insects). These species are effective predators of medically important pests such as snails, and mosquito larvae.     

Immunomarking reveals food flow and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Trophallaxis and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar), were examined using a novel marking technique, rabbit IgG protein coupled with an enzyme linked immunosorbent assay (ELISA) to detect the marker. Transfer experiments in small dishes evaluated the trophallactic transfer of the marker from donor workers fed IgG-treated paper to recipient workers or larvae. Worker donors rapidly acquired the marker, and 100% of donors tested positive within 24 h. Trophallactic transfer from donors to recipients was relatively inefficient, and 51 +/- 2% of recipient workers and 31 +/- 2% of recipient larvae tested positive at 72 h. Based on the mean optical density counts, approximately 27% of marker ingested by the donors was passed on to the recipient workers in the first 24 h, 14% to recipient larvae, and 26% to recipient soldiers. The ability of soldiers to feed independently of workers was examined in dish assays. Soldiers showed no significant uptake of the marker when isolated from the workers, and uptake increased significantly when workers were present. The distribution of the marker was further studied in larger colony fragments composed of workers, soldiers, nymphs, and larvae. Marker acquisition by the different castes/developmental stages was highly variable, with workers and nymphs acquiring the marker at a faster rate than soldiers and larvae. The results of this study contribute to our understanding of the foraging ecology and social behavior in R. flavipes. In addition, they may help design improved control programs for subterranean termites based on baits.     

Three new color patterns of nymphs of Arvelius albopunctatus (De Geer) (Hemiptera: Pentatomidae)

Arvelius albopunctatus (De Geer) is widely distributed in the Neotropical region, being an important pest of Solanaceae. Color variation of nymphs was described for this and other pentatomid species and their knowledge is essential for an early identification. From December 2005 to July 2006 nymphs and adults of A. albopunctatus were collected in forest fragments in Criciúma, I?ara, Maracajá and Araranguá (SC, Brazil). Known morphs of nymphs of both 4th and 5th instars were observed, as well as three new morphs, here described and named red morph (4th and 5th instars), green morph (4th and 5th instars) and orange morph (5th instar).     

亚洲柑橘木虱成虫和5龄若虫在感染黄龙病的柑橘上的取食行为及获菌效率比较

【目的】亚洲柑橘木虱Diaphorina citri是柑橘的毁灭性病害——黄龙病亚洲种‘Candidatus Liberibacter asiaticus’(‘C las’)的主要传播媒介。本研究的目的是明确木虱成虫和5龄若虫的取食行为、获菌效率是否有差异,以及寄主感染黄龙病是否对5龄若虫取食产生影响。【方法】利用直流型刺吸电位仪(DC-EPG Giga-4)记录柑橘木虱成虫和5龄若虫在携带黄龙病的酸橘Citrus reticulata cv.Sunki嫩梢上10 h的取食行为,用qPCR单头检测其获得黄龙病病原菌的效率,并比较5龄若虫在感病和健康植株嫩梢上的取食行为。【结果】柑橘木虱成虫与5龄若虫在感染黄龙病的酸橘上的取食行为有显著差异。5龄若虫比成虫更快地开始在韧皮部和木质部进行吸食,口针在韧皮部的总过程以及吸食时间显著长于成虫。此外,5龄若虫和成虫在EPG测定(同时饲菌)10 h后获菌率分别为37.5%和20.0%,若虫明显高于成虫。寄主植物感染黄龙病对5龄若虫的取食行为有一定的影响,表现在感病植株上的刺探次数、唾液分泌次数和韧皮部吸食次数都显著少于健康植株,而两者分泌唾液和韧皮部吸食时间没有显著差异。另外,在感病植株上首次韧皮部取食出现时间较在健康植株上要早。【结论】在同等时间下,柑橘木虱5龄若虫比成虫在感染黄龙病的酸橘上的取食能力更强、取食量更大、获菌率更高,原因可能是若虫需要更多的营养物质供其生长发育所致。寄主感染黄龙病对木虱5龄若虫的取食有利,使其更快地开始取食,而且更改取食位点次数变少,推测可能与黄龙病菌破坏了植物的防御有关。     

NMR and conformational studies of linear and cyclic oligo-(1→6)-β-D-glucosamines

The conformational behavior of a series of linear and cyclic oligo-(1→6)-β-D-glucosamines and their N-acetylated derivatives, which are related to fragments of natural poly-N-acetylglucosamine, was studied by theoretical molecular modeling and experimental determination of transglycosidic vicinal coupling constants (3)J(C,H) and (3)J(H,H). Molecular dynamics simulations were performed under several types of conditions varying in the consideration of ionization of amino groups, solvent effect, and temperature. Neural network clustering and asphericity calculations were performed on the basis of molecular dynamics data. It was shown that disaccharide fragments in the studied linear oligosaccharides were not rigid, and tended to have several conformers, thus determining the overall twisted shape with helical elements. In addition, it was found that the behavior of C5-C6 bond depended significantly upon the simulation conditions. The cyclic di-, tri-, and tetrasaccharides mostly had symmetrical ring-shaped conformations. The larger cycles tended to adopt more complicated shapes, and the conformational behavior of their disaccharide fragments was close to that in the linear oligosaccharides.     

p-Chloroamphetamine (PCA) suppresses ingestive behavior in male rats.

Ingestive behavior was activated in male rats by intraoral intake and intake from a bottle of 1-M solution of sucrose. Intraperitoneal injection of p-chloroamphetamine (PCA), releasing central 5-hydroxytryptamine (5-HT) from serotonergic nerve terminals, inhibited ingestion of the sucrose solution. Significant inhibition of sucrose intake by PCA was observed at 1.25 and 2.5 mg/kg dose in a bottle intake test, and at 5.0 mg/kg dose in an intraoral intake test. These findings suggested that 1.25 and 5.0 mg/kg of PCA suppressed appetitive ingestive behavior and consummatory ingestive behavior in male rats, respectively.     

Maternal behaviour of a webspinner (Order Embiidina)

ABSTRACT. 1. This first detailed behavioural field study of any species within the order Embiidina (=Embioptera) examines the function of parental care in the webspinner Clothoda urichi (Saussure) (Clothodidae).
2. Time budgets of nymphs and females in different stages of reproduction differ significantly due, in part, to maternal behaviours.
3. Nymphs, non-reproductive adult females and females with nymphs are active only at night when they feed and spin silk. In contrast, females with eggs guard their eggs both day and night, rarely feeding during the 6-week period prior to hatch.
4. Females physically repel egg parasites. In addition, females construct a substantial egg mass covering, of substrate materials and silk, which hinders parasites.     

Adaptation of the externally feeding bug <Emphasis Type="Italic">Elasmucha necopinata</Emphasis> (Hemiptera: Acanthosomatidae) to its fig host

Figs (Ficus) are keystone resources that maintain tropical biodiversity. Pollinators, non-pollinating fig wasps, and some insects that feed internally in syconia (the fruits of figs) synchronize their developmental stages with syconia. Other insects feed and develop externally on the syconia but little is known about adaptations in these insects. We investigated the life cycle of Elasmucha necopinata, a bug that develops externally on the syconia of Ficus hispida, a functionally dioecious fig. The bug oviposits about a week after fig receptivity, the nymphs feed externally on the syconia and eclosion occurs when the syconia mature. Thus, nymphs synchronize their developmental stages with the male syconia of F. hispida. Although the relationship does not appear to be mutualistic, we suggest that E. necopinata, feeding externally, has adapted to figs, in a similar way to Agaonid fig wasps that live internally. We believe that this is the first direct evidence of adaptation of an externally feeding insect to figs.     

Cyclization of the phosphorus chain of the methylene-bridged analogs of adenosine triphosphate.

The phosphorus-containing side chains of two methylene-bridged analogs of adenosine triphosphate have been cyclized to produce the corresponding analogs of monoadenosine-5'-trimetaphosphate. (The structures are given in the journal.) The molecules, which were generated in anhydrous media through a carbodimide-mediated condensation, were characterized by 31P nuclear-magnetic resonance, and the white-noise 1H decoupled spectra were simulated. These molecules are quite reactive and readily converted to their corresponding linear forms upon hydrolysis. The second structure contains an asymmetric phosphorus atom, and both of the possible cyclic molecules have been observed and the diasteroisomeric mixture has been isolated.