Disruption of insect immunity using analogs of the pleiotropic insect peptide hormone Neb-colloostatin: a nanotech approach for pest control II.

This work continues our studies on the pleiotropic activity of the insect peptide Neb-colloostatin in insects. In vivo immunological bioassays demonstrated that hemocytotoxic analogs of Neb-colloostatin injected into Tenebrio molitor significantly reduced the number of hemocytes in the hemolymph and impaired phagocytosis, nodulation and phenoloxidase activities in the insects. Among the analogs tested, [Ala1]-,[Val1]-, [Hyp4]- and [Ach4]-colloostatin were particularly potent in disrupting cellular immunity in larvae, pupae and adult insects. This result suggests that the most effective analogs showed increases in the bioactivity period in the hemolymph of insects when compared to Neb-colloostatin. Recently, we demonstrated that it is possible to introduce Neb-colloostatin through the cuticle of an insect into the hemolymph when the peptide is coupled with nanodiamonds. In this study, we showed that [Ala1]-, [Val1]-, [Hyp4]- and [Ach4]-colloostatin, when complexed with nanodiamonds, may also pass through the cuticle into the hemolymph and induce long-term impairments of immunity in T. molitor at all developmental stages. Studies on the tissue selectivity and effectiveness of Neb-colloostatin analogs and efficient methods for their introduction into insects may contribute to the development of eco-friendly pest control methods based on bioactive peptidomimetics.

Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells.

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.

Comparative psychoneuroimmunology: evidence from the insects.

Interactions between immune systems, nervous systems, and behavior are well established in vertebrates. A comparative examination of these interactions in other animals will help us understand their evolution and present adaptive functions. Insects show immune-behavioral interactions similar to those seen in vertebrates, suggesting that many of them may have a highly conserved function. Activation of an immune response in insects results in illness-induced anorexia, behavioral fever, changes in reproductive behavior, and decreased learning ability in a broad range of species. Flight-or-fight behaviors result in a decline in disease resistance. In insects, illness-induced anorexia may enhance immunity. Stress-induced immunosuppression is probably due to physiological conflicts between the immune response and those of other physiological processes. Because insects occupy a wide range of ecological niches, they will be useful in examining how some immune-behavioral interactions are sculpted by an animal's behavioral ecology.

Adipokinetic hormone and the immune responses of locusts to infection.

Injections of Bacillus, or of blastospores from the entomopathogenic fungus, Metarhizium anisopliae, activate the prophenoloxidase (PPO) cascade, and coinjection of adipokinetic hormone-I (AKH) enhances and prolongs these responses. When injected concurrently with an immunizing dose of live bacteria, AKH suppresses the appearance of antimicrobial activity and, after a short delay, increases the growth of bacteria within the hemocoel. Injections of live Escherichia coli or Pseudomonas aeruginosa into locusts fail to activate PPO in the hemolymph, even when coinjected with AKH. The coinjection of bacteria and hormone is rarely lethal to the locust. However, if locusts are injected with AKH when they are infected with Metarhizium, they die more rapidly than if no AKH is administered.

Induced hyperlipaemia and immune challenge in locusts.

Injections of immunogens, such as beta-1,3-glucan or lipopolysaccharide (LPS), bring about a marked hyperlipaemia with associated changes in lipophorins and apolipophorin-III in the haemolymph of Locusta migratoria. These changes are similar to those observed after injection of adipokinetic hormone (AKH). The possibility that endogenous AKH is released as part of the response to these immunogens is investigated using passive immunisation against AKH-I, and measurement of AKH-I titre in the haemolymph after injection of immunogens. The data presented show that, despite the similarity of the changes brought about by the presence of immunogens in the haemolymph to those brought about by AKH, there is no release of endogenous AKH after injection of laminarin or LPS. A direct effect of the immunogens on release of neutral lipids by the fat body cannot be demonstrated in vitro, and the mechanism by which hyperlipaemia is induced during immune challenge remains uncertain.

The cycling and distribution of PER-like antigen in relation to neurons recognized by the antisera to PTTH and EH in Thermobia domestica.

The cephalic nervous system of the firebrat contains antigens recognized by antisera to the clock protein period (PER), the prothoracicotropic hormone (PTTH) and the eclosion hormone (EH). The content of the 115 kDa PER-like antigen visualized on the western blots fluctuates in diurnal rhythm with a maximum in the night. The oscillations entrained in a 12:12 h light/dark (LD) cycle persist in the darkness and disappear in continuous light. They are detected by immunostaining in 14 pairs of the protocerebral neurons and are extreme in four suboesophageal neurons and two cells in each corpus cardiacum that contain PER only during the night phase. No circadian fluctuations occur in three lightly stained perikarya of the optic lobe. Five cell bodies located in each brain hemisphere between the deuto-and the tritocerebrum retain weak immunoreactivity under constant illumination. In all cells, the staining is confined to the cytoplasm and never occurs in the cell nuclei. The cells containing PER-like material do not react with the anti-PTTH and anti-EH antisera, which recognize antigens of about 50 and 20 kDa, respectively. The anti-PTTH antiserum stains in each brain hemisphere seven neurons in the protocerebrum, eight in the optic lobe, and 3-5 in the posterior region of the deutocerebrum. The antiserum to EH reacts in each hemisphere with just two cells located medially to the mushroom bodies. No cycling of the PTTH-like and EH-like antigens was detected.

Antibacterial peptides: basic facts and emerging concepts.

Antibacterial peptides are the effector molecules of innate immunity. Generally they contain 15-45 amino acid residues and the net charge is positive. The cecropin type of linear peptides without cysteine were found first in insects, whilst the defensin type with three disulphide bridges were found in rabbit granulocytes. Now a database stores more than 800 sequences of antibacterial peptides and proteins from the animal and plant kingdoms. Generally, each species has 15-40 peptides made from genes, which code for only one precursor. The dominating targets are bacterial membranes and the killing reaction must be faster than the growth rate of the bacteria. Some antibacterial peptides are clearly multifunctional and an attempt to predict this property from the hydrophobicity of all amino acid side chains are given. Gene structures and biosynthesis are known both in the fruit fly Drosophila and several mammals. Humans need two classes of defensins and the cathelicidin-derived linear peptide LL-37. Clinical cases show that deficiencies in these peptides give severe symptoms. Examples given are morbus Kostmann and atopic allergy. Several antibacterial peptides are being developed as drugs.

Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide.

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of LPS and is virtually complete within 8 h, nodules occurring mainly associated with the dorsal diaphragm on either side of the heart, but sometimes with smaller numbers associated with the ventral diaphragm on either side of the nerve cord. Co-injection of adipokinetic hormone-I (Lom-AKH-I) with LPS stimulates greater numbers of nodules to be formed in larval and adult locusts, and activates phenoloxidase in the haemolymph of mature adults but not of nymphs. The effect of co-injection of Lom-AKH-I with LPS on nodule formation is seen at low doses of hormone; only 0.4 pmol of Lom-AKH-I per adult locust is needed to produce a 50% increase in the number of nodules formed. When different components of LPS from the E. coli Rd mutant are tested, the mono- and the diphosphoryl Lipid A components have similar effects to the intact LPS. Remarkably, detoxified LPS activates phenoloxidase in the absence of Lom-AKH-I, although co-injection with hormone does enhance this response. Both diphosphoryl Lipid A and detoxified LPS induce a level of nodule formation that is enhanced by co-injection of Lom-AKH-I, but monophosphoryl Lipid A does not initiate nodule formation even when injected with hormone. Co-injection of a water-soluble inhibitor of eicosanoid synthesis, diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid), reduces nodule formation in response to injections of LPS (both in the absence and presence of hormone) in a dose-dependent manner, but does not prevent activation of phenoloxidase in adult locusts. It is shown that nodule formation and activation of the prophenoloxidase in locust haemolymph can both be enhanced by Lom-AKH-I, but it is argued that these processes involve distinct mechanisms in which eicosanoid synthesis is important for nodule formation, but not for the increased phenoloxidase activity.

Localization of crustacean hyperglycemic and vitellogenesis-inhibiting hormones in separate cell types in the protocerebrum of the woodlouse Armadillidium vulgare (Crustacea, Isopoda).

We gave an accurate immunolocalization of CHH (crustacean hyperglycemic hormone) and VIH (vitellogenesis-inhibiting hormone) in the brain and the sinus gland of the woodlouse Armadillidium vulgare. The two immune sera have been respectively raised against HPLC-purified CHH and against a small peptide derived from the N-terminus of VIH. By immunocytochemistry, we showed that CHH and VIH were synthesized in different perikarya and stored in different axon endings of the sinus gland. As in other crustacean species studied to date, CHH was located in the axon endings filled with the biggest granules. Immunoblotting confirmed that VIH was stored in the sinus glands of both the female and the male. These clear localizations of CHH- and VIH-antigens do not preclude that only one peptide is released from a given type of SG endings and do not rule out that each peptide can be involved in the control of different physiological processes.

Neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae.

Anatomical study of neurons projecting to the retrocerebral complex of the adult blow fly, Protophormia terraenovae, was done by NiCl2 filling and immunocytochemistry. Retrograde filling through the cardiac-recurrent nerve labeled three groups of neurons in the brain/subesophageal ganglion: (1) paramedial clusters of the pars intercerebralis, (2) neurons in each pars lateralis, and (3) neurons in the subesophageal ganglion. The pars intercerebralis neurons send prominent axons into the median bundle and exit from the brain via the contralateral nervus corporis cardiaci. Based on the projection pattern, two types of the pars lateralis neurons can be distinguished: the most lateral pairs of neurons contralaterally extend through the posterior lateral tract and the remainder ipsilaterally extend through the posterior lateral tract. The neurons in the subesophageal ganglion run through the contralateral nervus corporis cardiaci. The dendritic arborization of the pars intercerebralis and pars lateralis neurons is restricted to the superior protocerebral neuropil and to the anterior neuropil of the subesophageal ganglion where the neurons in the subesophageal ganglion also project. Retrograde filling from the corpus allatum indicated that the pars lateralis neurons and a few pars intercerebralis neurons project to the corpus allatum, but that the neurons in the subesophageal ganglion do not. Orthograde filling from the pars intercerebralis and staining by paraldehyde-thionin/paraldehyde-fuchsin indicated that the pars intercerebralis neurons project primarily to the corpus cardiacum/hypocerebral ganglion complex. Immunostaining with a polyclonal antiserum against diapause hormone, a member of the FXPRLamide family, suggests that some of the subesophageal ganglion neurons contain FXPRLamide-like peptides.

Neuropeptides in insect sensory neurones: tachykinin-, FMRFamide- and allatotropin-related peptides in terminals of locust thoracic sensory afferents.

Sensory afferents in the thoracic ganglia of the locust Locusta migratoria were labelled with antisera to different neuropeptides: locustatachykinins, FMRFamide and allatotropin. The locustatachykinin-immunoreactive (LTKIR) sensory fibres were derived from the legs and entered the ventral sensory neuropil of each of the thoracic ganglia via nerve 5. In the thoracic neuropil, the LTKIR sensory fibres formed a distinct plexus of terminations ventrally in the ipsilateral hemisphere. The peripheral cell bodies of the sensory neurones could not be revealed, but lesion experiments indicated that origin of the LTKIR fibres was the tarsus of each leg. Possibly the thin fibres are from tarsal chemoreceptors. Double labelling immunocytochemistry revealed that all the LTKIR sensory fibres contained colocalized FMRFamide immunoreactivity. A larger population of sensory fibres reacted with antiserum to moth (Manduca sexta) allatotropin. By means of double labelling immunocytochemistry, we could show that the LTKIR fibres constituted a subpopulation of the larger set of allatotropin-like immunoreactive fibres. Thus some sensory fibres may contain colocalized peptides related to locustatachykinins, FMRFamide-related peptide(s) and allatotropin-like peptide. A separate non-overlapping small set of sensory fibres in nerve 5 reacted with an antiserum to serotonin. Sensory fibres of the other nerves of the ventral nerve cord, including the abdominal ganglia, did not react with the peptide antisera. Since acetylcholine is the likely primary neurotransmitter of insect sensory fibres, it is possible that the peptides and serotonin are colocalized with this transmitter and serve modulatory functions in a subset of the leg afferents.

Postembryonic development of leucokinin-like immunoreactive neurons in the moth Spodoptera litura.

Antiserum to leucokinin I, a neuropeptide originally isolated from the cockroach Leucophaea maderae, was used for immunocytochemical labeling of neurons in the brain and ventral ganglia of the moth Spodoptera litura during postembryonic development. In the ventral ganglia, leucokinin-like immunoreactivity begins to occur in the abdominal ganglion A3 to A7 of first instar larva. One to two weakly labeled pairs of bilateral LK-LI cell bodies are located in the subesophageal ganglion of fourth to sixth instar larvae and in the abdominal ganglia A1 to A7 of second to sixth instar larvae. The abdominal ganglion A1 of fourth to sixth instar larvae and A8 of sixth instar larva each contain one weakly labeled pair of median LK-LI cell bodies. Two strongly labeled pairs of bilateral LK-LI neurons are found in A3 to A7 of third to sixth instar larvae. Abdominal ganglia A1 to A8 of prepupa, pupa and adult contain one to three weakly labeled pairs of bilateral LK-LI neurons. Two strongly labeled pairs of bilateral LK-LI neurons in each of the abdominal ganglia of larva, prepupa, pupa and adult send axons to the neuropil, and then each axon bifurcates into two axonal branches. Theses axonal branches from two bundles. From each of the two pairs of neurons an axon exits through the posterior ventral nerve (N2) which runs to the transverse nerve of the next posterior segment. In larval brains, 2-16 pairs of bilateral LK-LI cell bodies can be found together with LK-LI processes in the central neuropil. The larval brains show large changes in the number of LK-LI neurons throughout postembryonic development. The number of LK-LI cell bodies are reduced in number from sixth instar larval brain. Therefore, prepupal, pupal and adult brains contain a smaller number of LK-LI cell bodies. Two pairs of LK-LI median neurosecretory cells located immediately beside the pars intercerebralis in larval brains increase to three pairs in the 7-day-old pupal brain. In the adult, however, LK-LI median neurosecretory cells decrease to one pair.

Structures, assays and receptors for locust adipokinetic hormones.

This review is concerned mainly with the adipokinetic hormones (AKHs) of locusts: their molecular conformations, actions and functions and the development of microfiltration assays in vitro. The physiological significance of having multiple hormones with overlapping actions whose efficacy changes during development is discussed in relation to the possibility that these reflect variations in populations of receptors and/or the pharmacokinetics of the peptides. The involvement of second messengers in the transduction mechanism of AKHs is reviewed, and we describe hormone-induced changes of intracellular calcium in single dispersed fat body cells. The structure activity relationships of the three locust AKHs and a number of analogues with variations at the N- and C-termini are discussed. A number of areas are identified where there are gaps in our understanding of these hormones, and some of these will be the focus of our future research.

Conservation of hexamerin endocytosis in Diptera.

In cyclorrhaphan Diptera at least two different types of haemolymph proteins exist which belong to the class of hexamerins. In the last larval instar of Calliphora vicina, the highly aromatic hexamerin, arylphorin, and the second hexamerin, PII, make up about 90% of haemolymph proteins. Both of these proteins are selectively taken up by the fat body cells at the end of larval life and share a common membrane-bound receptor. In addition, hexamerins and possible hexamerin receptors of Calliphora vicina, Calliphora vomitoria, Drosophila melanogaster, Ceratitis capitata, Sarcophaga bullata, Musca domestica and Protophormia terraenovae were investigated. Uptake of arylphorin by the larval fat bodies of Calliphora vicina as well asarylphorin-receptor binding can be competed in vitro by haemolymph from other Diptera. Therefore, hexamerin-receptor binding must be conserved among related cyclorrhaphan Diptera and between different types of hexamerins within a species. As the degree of competition is in good agreement with the presumed phylogenetic distances between these species, the method described here provides a simple tool to estimate evolutionary distances.

Drosophila ribosomal protein PO contains apurinic/apyrimidinic endonuclease activity.

Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to generate polyclonal antibodies for Western analysis. Biochemical tests were originally performed to see if overexpressed PO contained DNase activity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3. The overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3. As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glutathione S-transferase (GST) fusions containing a Factor Xa cleavage site between GST and PO were constructed, overexpressed in an E.coli strain defective for the major 5'-acting AP endonucleases and the fusions purified using glutathione-agarose affinity column chromatography. Isolated fractions containing purified GST-PO fusion proteins were subsequently found to have authentic AP endonuclease activity. Moreover, glutathione-agarose was able to deplete AP endonuclease activity from GST-PO fusion protein preparations, whereas the resin was ineffective in lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage. These results suggested that PO was a multifunctional protein with possible roles in DNA repair beyond its known participation in protein translation. In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonucleases from sensitivity to an alkylating agent. We furthermore show that GST-PO can be located in both the nucleus and ribosomes. Its nuclear location can be further traced to the nuclear matrix, thus placing PO in a subcellular location where it could act as a DNA repair protein. Other roles beyond DNA repair seem possible, however, since GST-PO also exhibited significant nuclease activity for both single- and double-stranded DNA.

Ovarian and hemolymph ecdysteroids in the terrestrial isopod Armadillidium vulgare (Malacostracan Crustacea).

Ovarian and hemolymph ecdysteroids in Armadillidium vulgare were analyzed at four stages of ovarian maturation through the reproductive molt cycle using high-performance liquid chromatography and radioimmunoassay. The major ecdysteroids in the ovaries and hemolymph of A. vulgare were 20-hydroxyecdysone and ecdysone in free and conjugated forms. The concentration of ovarian ecdysteroids reached a maximal level in maturing ovaries during stage D (premolt period) of the molt cycle. At the end of stage D, a high level of ecdysteroids was detected in fully matured ovaries. On the other hand, hemolymph ecdysteroid titers in reproductive females showed a peak during stage D and declined rapidly to a low level at the end of stage D. No appreciable differences in the amounts of hemolymph ecdysteroids and in molecular species of them were apparent in females in reproductive and nonreproductive molt cycles. The amounts of hemolymph ecdysteroids were about fivefold higher in females than those in males. These ecdysteroids may have a role in controlling ovarian development in female A. vulgare.

Immunolocalization of the oostatic and prothoracicostatic peptide, Neb-TMOF, in adults of the fleshfly, Neobellieria bullata.

The hexapeptide Neb-TMOF (H-NPTNLH-OH, trypsin modulating oostatic factor of the gray fleshfly, Neobellieria bullata)2 occurs in vitellogenic ovaries and is involved in negative feedback regulation of trypsin biosynthesis in the gut of late vitellogenic females. Polyclonal antisera were raised against the synthetic peptide and were used to identify and immunolocalize Neb-TMOF epitopes in different fleshfly tissues. Neb-TMOF-immunoreactive material first appears in the cortical layer of young vitellogenic oocytes and later spreads over the yolk granules. This suggests a pinocytosis with the three yolk polypeptides (vitellogenins). Controls treated with the preimmune sera or with anti-Neb-TMOF antiserum preadsorbed to Neb-TMOF peptide coupled to a solid phase support did not stain. There was no immunostaining in the central nervous system (brain and ventral nerve cord), the retrocerebral complex, the fat body, or the testes. Western blot analysis showed that the anti-Neb-TMOF antisera specifically recognize a putative hormone precursor polypeptide (Mr 75 kDa) from vitellogenic ovaries. This protein is virtually absent from the hemolymph. It is not immunologically related to the three yolk polypeptides, since it is not recognized by yolk polypeptides antisera. In adult females the ovary appears to be the only site of synthesis of Neb-TMOF and of its precursor. Immunopositive staining is found in the apical areas of ovarian follicle cells, suggesting these cells as a site of hormone precursor biosynthesis. This is the first demonstration that a protein colocalized with yolk proteins might act as a precursor for a folliculostatic hormone.