A comparison between the amino acid composition of an egg parasitoid wasp and some of its hosts

Amino acid compositions of the eggs of five lepidopteran hosts for Trichogramma minutum were compared with each other and with a non-host species, Rhodnius prolixus, in which T. minutum oviposits but does not develop. Host eggs are quite homogeneous, particularly when compared according to groupings of potentially interconvertible amino acids. Combined mole percent values for glycine, serine and alanine were higher in hosts (27.5–29.2 mole%) than in R. prolixus eggs (21.5 mole%), in bovine serum albumin (14.9%), which has been used as a protein source in artificial diets for T. minutum, or in many of the mixtures used in published diets for this species. Since these three amino acids make up 26.3 mole% of the adult amino acid content of T. minutum, their deficiency in diets could require metabolic compensation detrimental to development.Adult T. minutum arising from eggs of Manduca sexta, Choristoneura fumiferana, and Sitotroga cerealella are similar in amino acid composition to each other and, in general, to their hosts. Variability appears greater in hosts than in adult wasp composition, suggesting some interconversion of host amino acids to accommodate inflexible nutritional requirements of T. minutum.In the three host species tested, free amino acids constituted 15.8–19.3% by weight of the amino acid in egg contents. In M. sexta eggs, glycine, serine and alanine together make up 28.4% by weight of the total free amino acid, a much higher proportion than in many published diets. The four free amino acids (isoleucine, leucine, phenylalanine and histidine) reported to be oviposition stimulants in experiments on encapsulated diets are present in sufficient concentrations to induce oviposition in the host species tested and in R. prolixus. S. cerealella egg contents having approximately 1.8 g amino acid, yield one or rarely two adult T. minutum (1g amino acid/insect). In contrast, M. sexta eggs with 94 g amino acid each yield an average of 10–12 adults (8.2g amino acid/insect). This suggests that small hosts are allocated few eggs which can only develop into small adults because of nutrient supply (parasitoid size in metabolically restricted), whereas much larger hosts are allocated proportionately fewer eggs than the former resulting in larger, and presumably more viable and fecund, adults (parasitoid size is established behaviourally).     

Neuropeptide F and the corn earworm, Helicoverpa zea: A midgut peptide revisited

The neuropeptide Y family of peptides is implicated in the regulation of feeding across a broad range of animals, including insects. Among vertebrates, neuropeptide Y exerts its actions mainly centrally, whereas peptide YY and pancreatic polypeptide arise from digestive tissues. Among invertebrates, neuropeptide F (NPF) is the sole counterpart of the NPY family. Shared features of NPF sequences derived for Lepidoptera indicate that the midgut peptide (Hez-MP-I) of the corn earworm, Helicoverpa zea, characterized more than a decade ago, is a carboxyl fragment of a full-length NPF. An antibody to Hez-MP-I was used to characterize the peptide's distribution in tissues of larvae, pupae, and adults. Immunostaining demonstrated NPF-related material both in nervous tissues and in abundant endocrine cells of the midgut. Radioimmunoassay of Hez-MP-I in the head, midgut and hemolymph of fifth instar larvae revealed concentration changes corresponding to development and feeding state. As with the vertebrate homologs, NPF may arise both centrally and peripherally to modulate the physiology of feeding and digestion of Lepidoptera.     

Characterization of neuropeptide F-like immunoreactivity in the blood-feeding hemipteran, Rhodnius prolixus

The invertebrate neuropeptide Y (NPY) homolog, neuropeptide F (NPF), has been characterized for a wide range of invertebrate phyla, including platyhelminthes, molluscs, and arthropods. Current hypotheses suggest that NPF may be capable of regulating responses to diverse external cues related to nutritional status and feeding. The qualitative and quantitative distribution of an NPF-like peptide in fifth instar Rhodnius prolixus was undertaken using an antiserum raised against Drosophila NPF. Immunohistochemistry reveals NPF-like immunoreactive neurons and processes in the central nervous system, stomatogastric nervous system and peripheral nervous system. The distribution of NPF-like immunoreactivity within the medial neurosecretory cells of the brain and neurohemal areas of the corpus cardiacum and dorsal vessel, suggests NPF may act as a neurohormone. Immunoreactive processes are present over the surface of the hindgut and the immunoreactivity in these processes is greatly reduced in intensity 24h post-feeding. The quantification of partially purified NPF-like material in the CNS of R. prolixus was conducted by HPLC fractionation and radioimmunoassay. The results suggest that NPF-like material is present in fifth instar R. prolixus and likely released into the hemolymph following a blood meal.     

Neuropeptide F signaling regulates parasitoid-specific germline development and egg-laying in Drosophila

Drosophila larvae and pupae are at high risk of parasitoid infection in nature. To circumvent parasitic stress, fruit flies have developed various survival strategies, including cellular and behavioral defenses. We show that adult Drosophila females exposed to the parasitic wasps, Leptopilina boulardi, decrease their total egg-lay by deploying at least two strategies: Retention of fully developed follicles reduces the number of eggs laid, while induction of caspase-mediated apoptosis eliminates the vitellogenic follicles. These reproductive defense strategies require both visual and olfactory cues, but not the MB247-positive mushroom body neuronal function, suggesting a novel mode of sensory integration mediates reduced egg-laying in the presence of a parasitoid. We further show that neuropeptide F (NPF) signaling is necessary for both retaining matured follicles and activating apoptosis in vitellogenic follicles. Whereas previous studies have found that gut-derived NPF controls germ stem cell proliferation, we show that sensory-induced changes in germ cell development specifically require brain-derived NPF signaling, which recruits a subset of NPFR-expressing cell-types that control follicle development and retention. Importantly, we found that reduced egg-lay behavior is specific to parasitic wasps that infect the developing Drosophila larvae, but not the pupae. Our findings demonstrate that female fruit flies use multimodal sensory integration and neuroendocrine signaling via NPF to engage in parasite-specific cellular and behavioral survival strategies.     

Studies in the biochemistry of cirripede eggs. IV. The free Amino-acid pool in the eggs of Balanus balanoides (L.) and B. balanus (L.) during development

Changes in the ‘free’ amino acids, betaine, and trimethylamine oxide during the development of the eggs of Balanus balanoides (L.) and B. balanus (L.) have been determined; the results are given in terms of μM/g dry wt, μM/g water, and μM/10⁶ eggs. The amino acids are derived from the yolk proteins the net composition of which is known. Free amino acids are present in considerable quantity, as is commonly the case with crustacean tissue. Changes in the individual amino acids are discussed. B. balanus eggs contain large, and relatively constant, amounts of sarcosine; its function is unknown but large quantities are present in the more highly evolved cirripedes so far examined. A possible relation between betaine glycine, and sarcosine relative to choline metabolism is considered. Large amounts of taurine are present. There is a striking increase in β-alanine in the late stages of development; in B. balanoides it comes to be the most, and in B. balanus the third most common amino acid; its possible involvement in purine metabolism is considered. The relation between the amounts of the various entities in the eggs and in the bodies of the adult are examined.     

The pattern of neuropeptide F and RF-amide in two tapeworms

Two years ago the first platyhelminth regulatory peptide, neuropeptide F (NPF), was isolated from the tapeworm Moniezia expansa by Maule et al. (1991). NPF is a 39 amino acid peptide with a C terminal phenylalaninamide. NPF is the first platyhelminth neuropeptide to be sequenced fully. Preabsorption with NPF quenches the immunostaining with anti-FMRF-amide and anti-bovine PP (Halton et al. 1992). As the first authentic flatworm neuropeptide, the occurence and distribution of NPF along the whole flatworm line are under investigation. Both free-living and parasitic flatworms are being studied. So far NPF-immunoreactivity has been reported from three free- living flatworms (see Grahn et al., 1995) and from four parasitic flatworms (Marks et al., 1993).FMRF- and RF-amide immunoreactive (IR) nerve cells and fibres are common in the gull-tapeworm Diphyllobothrium dendriticum. In order to test whether the patterns for NPF- and RF-immunoreactivity co- localize in the gull-tapeworm, immunostaining with anti-NPF and anti-RF were performed. To broaden the study, adult Proteochepalus exiguus from the intestine of whitefish were included in the experiment.The study was performed on whole mounts of skinned worms (Gustafsson, 1991). Anti-NPF was used in concentrations 1:500 and 1:1000. Controls included liquid phase absorption with the homologous antigen (1000 ng ml^–1).In D. dendriticum NPF-immunoreactivity occurs in nerve cells and varicose nerve fibres of larval and adult worms. The NPF-IR cell bodies are more common in the peripheral nerve cords than along the main nerve cords, which contain nerve fibres with large varicosities. The cell bodies in the PNS are often triangular in shape. Immediately beneath the tegumental surface a thin NPF-IR nerve fibre is observed. As to the co-localization of NPF and RF nothing definite can be said but the general pattern seems tobe the same. In the brain commissure of D. dendriticum one large ganglion cell stains with both antisera, indicating coexistence.In P. exiguus NPF- and RF-immunoreactivity was observed in the two main nerve cords situated laterally and in the pairs of thin dorsal and ventral longitudinal nerve cords. Numerous transverse commissures connect the longitudinal cords forming an orthogonal pattern. The cell bodies along the nerve cords are multipolar. Thin projections extend from the main nerve cords to the surface of the worm. The main nerve cords are lined with NPF-and RF-IR cell bodies. The general staining patterns of NPF and RF are very similar.     

Expanding abdominal cuticle in the bug Rhodnius and the tick Boophilus

The abdominal cuticles of Rhodnius prolixus (fifth instar) and Boophilus microplus (adult female) expand dramatically and rapidly during feeding. In the unfed stage of both species the epicuticle of the abdomen is deeply folded, and when rapid stretching takes place the epicuticle unfolds and the underlying procuticle stretches so that the thickness of the cuticle is halved. The cuticles contained only trace amounts of protein soluble in water and aqueous KCl but substantial quantities were extracted with 7 M aqueous urea. The proteins were analysed for their amino acid composition and investigated by gel electrophoresis and isoelectric focusing.In solubility, amino acid composition, molecular weight distribution, and isoelectric points, the proteins isolated from both species resembled one another closely. They had higher molecular weights and higher isoelectric points than did the proteins from flexible, non-stretching cuticles and unlike them had high alanine and histidine and low aspartic acid and glutamic acid contents. Their amino acid composition was very similar to that of the whole cuticle. The proteins were not associated with neutral sugars. Both the Rhodnius and Boophilus cuticles had low chitin contents, 11·2 and 3·8% respectively (on a water-free basis). The composition of the cuticles and the properties of the proteins are discussed in relation to the stretching which they undergo.     

Neuropeptide F and its expression in the yellow fever mosquito,Aedes aegypti

A neuropeptide F (NPF) was isolated from an extract of adult Aedes aegypti mosquitoes based on its immunoreactivity in a radioimmunoassay for Drosophila NPF. After sequencing the peptide, cDNAs encoding the NPF were identified from head and midgut. These cDNAs encode a prepropeptide containing a 36 amino acid peptide with an amidated carboxyl terminus, and its sequence shows it to be a member of the neuropeptide F/Y superfamily. Immunocytochemistry and Northern blots confirmed that both the brain and midgut of females are likely sources of NPF, found at its highest hemolymph titer before and 24 h after a blood meal.     

Identification of kinin-related peptides in the disease vector, Rhodnius prolixus

We have used an in silico approach to identify a gene from the blood-gorging vector, Rhodnius prolixus, that is predicted to produce an insect kinin prepropeptide. The prepropeptide is 398 amino acids in length and can potentially produce a large number of kinin-related peptides following post-translational processing. A comparison with other insect kinin precursor sequences demonstrates greatest conservation at the C-terminal region of the kinin peptides. Multiple peptides predicted from the kinin gene are phenotypically expressed in R. prolixus, as revealed by MALDI-TOF MS MS, including 12 kinins and one kinin precursor peptide (KPP). Six of these peptides are characterized by the typical insect kinin C-terminal motif FX₁X₂WGamide and five of these are also found as truncated forms. Five peptides were identified with an atypical, though similar, FX₁X₂WAamide C-terminus. There is also peptide with a C-terminal DDNGamide motif and a number of non-amidated peptides.     

Female odours promote the activation of sheltered kissing bug Rhodnius prolixus males and modulate their orientation

The existence of a pheromone emitted during copulation has been reported for Rhodnius prolixus (Hemiptera: Reduviidae) Stål. Adults possess one pair of metasternal glands (MGs) from which female R. prolixus release volatiles mainly at night. We investigated whether these volatiles emitted by adult R. prolixus can modulate sexual‐related behaviours of opposite and/or same sex individuals. We first used a shelter bioassay to test if adult activity patterns can be affected by chemical signals emitted by opposite sex conspecifics. We observed that males left the shelter more frequently in the presence of females, showing higher activity and an increase of intrasexual copulation attempts. Females showed no shelter‐related activation in the presence of male odours. Second, we used a locomotion compensator device to investigate whether females or males show oriented responses to odours emitted by adults. We found that males oriented towards air currents carrying female odours but females did not orient towards odours emitted by adults. Finally, we observed that males oriented towards the female MG compounds. Thus, a volatile chemical signal emitted by females from their MGs promoted the activation of sheltered males and modulated orientation to air currents.     

Sulfated glycosaminoglycans in two hematophagous arthropod vectors of Chagas disease, Triatoma brasiliensis and Rhodnius prolixus (Hemiptera: Reduviidae)

The characterization of sulfated glycosaminoglycans (GAGs) in hematophagous arthropod vectors in general has been limited, with the exception of the studies in the triatomine Rhodnius prolixus. Heparan sulfate (HS) and chondroitin sulfate (CS) were previously identified and structurally characterized in extracts of whole bodies of fourth instar larvae of R. prolixus. Recently, we showed the expression of these two sulfated GAGs in specific body tissues of adult males and females and in embryos of R. prolixus. In the present work, we identified and compared the sulfated GAG composition in specific tissues of adult insects and in embryos of another triatomine species, Triatoma brasiliensis. Sulfated GAGs were isolated from the fat body, intestinal tract, and the reproductive tracts of adult insects and from embryos. Only HS and CS were found in the tissues analyzed. The present results extend the initial observations on the sulfated GAG composition in R. prolixus by showing that these molecules are widely distributed among internal organs of triatomines. These observations may be useful for future investigations aiming to evaluate the possible implication of these compounds in physiological events that take place in a specific organ(s) in these insects.     

Females of a blood-sucking bug may adjust their mating decisions according to the risk of ovipositing infertile eggs

Females should adjust their mating preferences when the costs of being selective vary. Here, we focus on the cost of laying unfertilized eggs. Oviparous species lose their fertilizable oocytes if remain virgin for too long. Thus, females who are at a high risk of laying infertile eggs should be selected against rejecting a mating opportunity under such circumstances. We test this prediction using the blood-sucking bug Rhodnius prolixus females, which exhibit stereotyped discrimination behaviour to undesired copula. Under laboratory conditions, we compared the copulatory rejection behaviour between females at their first ovarian cycle (12 days old adults, at low risk of ovipositing infertile eggs) and females at their second ovarian cycle (26 days old adults, at high risk of ovipositing infertile eggs). Since R. prolixus female rejection behaviour is mainly exhibited in the presence of potential mates, we also manipulated the presence and absence of additional males. We found that rejection behaviour significantly decreases both when females enter their second ovarian cycle (and are at high risk of losing unfertilized eggs) and under the absence of additional males. We discuss the possible effects of the risk of losing fertilizable eggs on female mating preferences.     

Discovery of multiple neuropeptide families in the phylum Platyhelminthes

Available evidence shows that short amidated neuropeptides are widespread and have important functions within the nervous systems of all flatworms (phylum Platyhelminthes) examined, and could therefore represent a starting point for new lead drug compounds with which to combat parasitic helminth infections. However, only a handful of these peptides have been characterised, the rigorous exploration of the flatworm peptide signalling repertoire having been hindered by the dearth of flatworm genomic data. Through searches of both expressed sequence tags and genomic resources using the basic local alignment search tool (BLAST), we describe 96 neuropeptides on 60 precursors from 10 flatworm species. Most of these (51 predicted peptides on 14 precursors) are novel and are apparently restricted to flatworms; the remainder comprise nine recognised peptide families including FMRFamide-like (FLPs), neuropeptide F (NPF)-like, myomodulin-like, buccalin-like and neuropeptide FF (NPFF)-like peptides; notably, the latter have only previously been reported in vertebrates. Selected peptides were localised immunocytochemically to the Schistosoma mansoni nervous system. We also describe several novel flatworm NPFs with structural features characteristic of the vertebrate neuropeptide Y (NPY) superfamily, previously unreported characteristics which support the common ancestry of flatworm NPFs with the NPY-superfamily. Our dataset provides a springboard for investigation of the functional biology and therapeutic potential of neuropeptides in flatworms, simultaneously launching flatworm neurobiology into the post-genomic era.     

A photosensitive circadian oscillator in an insect endocrine gland: photic induction of rhythmic steroidogenesis in vitro

The paired prothoracic glands of the insect Rhodnius prolixus each comprise a group of about 200 structurally identical cells. The synthesis (and release) of steroid moulting hormones (ecdysteroids) by these glands is under circadian control in vivo. We monitored ecdysteroid synthesis by single glands during long-term incubations in vitro. Synthesis is rhythmic in vitro and persists in continuous darkness. Glands which are arrhythmic (from prolonged continuous light) respond to transfer to darkness in vitro with the initiation of a free-running circadian rhythm of ecdysteroid synthesis. Therefore, the glands possess a light-sensitive circadian oscillator. These properties are conventionally associated with nervous tissue of animals. It is suggested that rhythmicity is synchronized within the gland by the known structural and electrical coupling between its component cells. The glands share properties with known pacemakers such as the avian pineal. However, the glands in vivo receive input from both light cues and the cerebral neuropeptide, prothoracicotropic hormone. Rhythmic release of this neuropeptide is controlled by a second oscillator located in the brain. We conclude that the pacemaker in the endocrine system of R. prolixus comprises at least three oscillators, one in each prothoracic gland and one in the brain, which are coupled hormonally. We conclude that the prothoracic gland is an important component of the circadian system controlling development in R. prolixus and that peripheral endocrine glands may play a more active role in the generation of animal circadian organization than has been thought. Accepted: 30 August 1997     

Structure and bioactivity of neuropeptide F from the human parasites Schistosoma mansoni and Schistosoma japonicum

The blood flukes Schistosoma mansoni and Schistosoma japonicum inflict immense suffering as agents of human schistosomiasis. Previous investigations have found the nervous systems of these worms contain abundant immunoreactivity to antisera targeting invertebrate neuropeptide Fs (NPFs) as well as structurally similar neuropeptides of the mammalian neuropeptide Y (NPY) family. Here, cDNAs encoding NPF in these worms were identified, and the mature neuropeptides from the two species differed by only a single amino acid. Both neuropeptides feature the characteristics common among NPFs; they are 36 amino acids long with a carboxyl-terminal Gly-Arg-X-Arg-Phe-amide and Tyr residues at positions 10 and 17 from the carboxyl terminus. Synthetic S. mansoni NPF potently inhibits the forskolin-stimulated accumulation of cAMP in worm homogenates, with significant effects at 10(-11) m. This is the first demonstration of an endogenous inhibition of cAMP by an NPF, and because this is the predominant pathway associated with vertebrate NPY family peptides, it demonstrates a conservation of downstream signaling pathways used by NPFs and NPY peptides.     

Polyamine Synthesis by the Mermithid Nematode Romanomermis culicivorax

The polyamine and amino acid composition of the mermithid nematode, Romanomermis culicivorax, and its host, Aedes aegypti, was determined. Putrescine, spermidine, spermine, cadaverine and two acetylated spermidine derivatives were present in parasitic juveniles, newly-emerged post-parasites, and eggs of R. culicivorax. Whole insect homogenates of fourth-instar A. aegypti contained the same array of polyamines, except that the putrescine:spermidine ratio was the inverse of that in parasitic R. culicivorax. Polyamines and amino acids were in greater concentrations in the nematode eggs than in other developmental stages investigated. Both the host and nematode possess the biosynthetic capacity for polyamine biosynthesis, as evidenced by measurable activities of ornithine decarboxylase in the host''s tissues and the nematode''s free-living stages.