PHARMACOLOGICAL REGULATION OF DIGESTION IN THE ANAUTOGENOUS FLESH FLY, Sarcophaga crassipalpis, BY SIMPLE INJECTION OF 6-HYDROXYDOPAMINE.

Female anautogenous Sarcophaga flesh flies need a protein meal to start large-scale yolk polypeptides (YPs) production and oocyte maturation. Protein meal rapidly elicits a brain-dependent increase in midgut proteolytic activity. Trypsin and chymotrypsin together represent over 80% of protease activity in liver-fed flies. Abdominal injection of 6-hydroxydopamine (6-OHDA) dose-dependently prohibits this increase in proteolytic activity at translational level in a similar way as post liver feeding decapitation. Delayed injection of 6-OHDA later than 6 h post liver meal has no effect. In flesh flies, chemical decapitation by 6-OHDA, by interrupting the brain-gut dopaminergic signaling, can be used as tool for the controlled inhibition of midgut proteolytic activity and subsequent ovarial development. Inhibition of ovarial development is probably indirect due to a deficit in circulating amino acids needed for YPs synthesis.

A quantitative peptidomics approach to unravel immunological functions of angiotensin converting enzyme in Locusta migratoria.

Locusta migratoria angiotensin converting enzyme (LmACE) is encoded by multiple exons displaying variable number of genomic duplications. Treatments of lipopolysaccharide (LPS) as well as peptidoglycan but not ß-1-3 glucan resulted in enhanced expression of angiotensin converting enzyme in hemocytes of Locusta migratoria. No such effect was observed in fat body cells. Differential peptidomics using locust plasma samples post infection with LPS in combination with both an LmACE transcript knockdown by RNAi and a functional knockdown using captopril allowed the identification of 5 circulating LPS induced peptides which only appear in the hemolymph of locust having full LmACE functionality. As these peptides originate from larger precursor proteins such as locust hemocyanin-like protein, having known antimicrobial properties, the obtained results suggest a possible direct or indirect role of LmACE in the release of these peptides from their precursors. Additionally, this experimental setup confirmed the role of LmACE in the clearance of multiple peptides from the hemolymph.

The endocrine system controlling sexual reproduction in animals: Part of the evolutionary ancient but well conserved immune system?

Drastic changes in hormone titers, in particular of steroid hormones, are intuitively interpreted as necessary and beneficial for optimal functioning of animals. Peaks in progesterone- and estradiol titers that accompany the estrus cycle in female vertebrates as well as in ecdysteroids at each molt and during metamorphosis of holometabolous insects are prominent examples. A recent analysis of insect metamorphosis yielded the view that, in general, a sharp rise in sex steroid hormone titer signals that somewhere in the body some tissue(s) is undergoing programmed cell death/apoptosis. Increased steroid production is part of this process. Typical examples are ovarian follicle cells in female vertebrates and invertebrates and the prothoracic gland cells, the main production site of ecdysteroids in larval insects. A duality emerges: programmed cell death-apoptosis is deleterious at the cellular level, but it may yield beneficial effects at the organismal level. Reconciling both opposites requires reevaluating the probable evolutionary origin and role of peptidic brain hormones that direct steroid hormone synthesis. Do e.g. Luteinizing Hormone in vertebrates and Prothoracicotropic Hormone (PTTH: acting through the Torso receptor) in insects still retain an ancient role as toxins in the early immune system? Does the functional link of some neuropeptides with Ca(2+)-induced apoptosis make sense in endocrine archeology? The endocrine system as a remnant of the ancient immune system is undoubtedly counterintuitive. Yet, we will argue that such paradigm enables the logical framing of many aspects, the endocrine one inclusive of both male and female reproductive physiology.

Differential peptidomics highlights adipokinetic hormone as key player in regulating digestion in anautogenous flesh fly, Sarcophaga crassipalpis.

Females of anautogenous flesh flies, Sarcophaga crassipalpis, need a protein meal in order to produce their first batch of eggs. This protein meal elicits an increase in midgut proteolytic activity that is under neuropeptidergic regulation. Time series of decapitation and rescue experiments of liver fed flies evidenced the need of a peptide factor released by corpora cardiaca (CC) within 4h post protein feeding in order to assure complete protein digestion. Q-Exactive quantitative differential peptidomics analysis on CC of sugar fed flies and flies 5h post protein feeding respectively, showed a unique consistent decrease in the stored amount of adipokinetic hormone (AKH) ranging between 16% up to 63%. Injection of AKH into liver fed decapitated flies as well as sugar fed intact flies resulted in dose dependent enhanced midgut proteolytic activity up to the level of intact protein fed flies. This suggests a key role of AKH in food depended reproduction.

Characterisation of a functional allatotropin receptor in the bumblebee, Bombus terrestris (Hymenoptera, Apidae).

Allatotropins (ATs) are multifunctional neuropeptides initially isolated from the tobacco hornworm, Manduca sexta, where they were found to stimulate juvenile hormone synthesis and release from the corpora allata. ATs have been found in a wide range of insects, but appear to be absent in Drosophila. The first AT receptor (ATR) was characterised in 2008 in the lepidopteran Bombyx mori. Since then ATRs have been characterised in Coleoptera and Diptera and in 2012, an AT precursor gene was identified in hymenopteran species. ATRs show large sequence and structural similarity to vertebrate orexin receptors (OXR). Also, AT in insects and orexin in vertebrates show some overlap in functions, including modulation of feeding behaviour and reproduction. The goal of this study was to identify a functional ATR in a hymenopteran species. We used ATRs (insect sequences) and OXRs (vertebrate sequences) to search the genome of the bumblebee, Bombus terrestris. Two receptors (XP_003402490 and XP_003394933) with resemblance to ATRs and OXRs were found. Phylogenetic analysis provided the first indication that XP_003402490 was more closely related to ATRs than XP_003394933. We investigated the transcript level distribution of both receptors and the AT precursor gene by means of quantitative real-time reverse transcriptase PCR. XP_003402490 displayed a tissue distribution comparable with ATRs in other species, with high transcript levels in the male accessory glands. After pharmacological characterisation, it appeared that XP_003402490 is indeed a functional ATR. Activation of the receptor causes an increase in intracellular calcium and cyclic AMP levels with an EC50 value in the low nanomolar to picomolar range. XP_003394933 remains an orphan receptor.

Age- and task-dependent foraging gene expression in the bumblebee Bombus terrestris.

In eusocial insects, the division of labor within a colony, based on either age or size, is correlated with a differential foraging (for) gene expression and PKG activity. This article presents in the first part a study on the for gene, encoding a cGMP-dependent protein kinase (PKG) in the bumblebee Bombus terrestris. Cloning of the open reading frame allowed phylogenetic tracing, which showed conservation of PKGs among social insects. Our results confirm the proposed role for PKGs in division of labor. Btfor gene expression is significantly higher in the larger foragers compared with the smaller sized nurses. More importantly, we discovered an age-related decrease in Btfor expression in both nursing and foraging bumblebees. We therefore speculate that the presence of BtFOR is required for correct adaptation to new external stimuli and rapid learning for foraging. In a second series of experiments, worker bumblebees of B. terrestris were treated with two insecticides imidacloprid and kinoprene, which have shown to cause impaired foraging behavior. Compared with controls, only the latter treatment resulted in a decreased Btfor expression, which concurs with a stimulation of ovarian growth and a shift in labor toward nest-related tasks. The data are discussed in relation to Btfor expression in the complex physiological event of foraging and side-effects by pesticides.

Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions.

BACKGROUND: To obtain reliable quantitative RT-PCR data, normalization relative to stable housekeeping genes is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in insects are extremely rare and have never been performed in locusts. In this study, putative housekeeping genes were identified in the desert locust, Schistocerca gregaria and two different software programs (geNorm and Normfinder) were applied to assess the stability of these genes. RESULTS: We have identified seven orthologs of commonly used housekeeping genes in the desert locust. The selected genes were the orthologs of actin, EF1a, GAPDH, RP49, TubA1, Ubi, and CG13220. By employing real time RT-PCR we have analysed the expression of these housekeeping genes in brain tissue of fifth instar nymphs and adults. In the brain of fifth instar nymphs geNorm indicated Sg-EF1a, Sg-GAPDH and Sg-RP49 as most stable genes, while Normfinder ranked Sg-RP49, Sg-EF1a and Sg-ACT as most suitable candidates for normalization. The best normalization candidates for gene expression studies in the brains of adult locusts were Sg-EF1a, Sg-GAPDH and Sg-Ubi according to geNorm, while Normfinder determined Sg-GAPDH, Sg-Ubi and Sg-ACT as the most stable housekeeping genes. CONCLUSION: To perform transcript profiling studies on brains of the desert locust, the use of Sg-RP49, Sg-EF1a and Sg-ACT as reference genes is proposed for studies of fifth instar nymphs. In experiments with adult brains, however, the most preferred reference genes were Sg-GAPDH, Sg-Ubi and Sg-EF1a. These data will facilitate transcript profiling studies in desert locusts and provide a good starting point for the initial selection of genes for validation studies in other insects.

Sublethal effects of crop protection on honey bee pollination: foraging behaviour and flower visits.

Crop protection strategies essential for pest and disease control can pose risk to pollinators. Fruits cannot be grown commercially without the use of crop protection agents, either from organic or chemical origin. The use of products with toxic effects is banned during flowering, and precise pre-flowering intervals have to be respected in Good Agricultural Practice. Bee pollination is essential for fruit crops to guarantee maximal fruit quality (shape and size) and quantity (yield weight). Fruit growers in Belgium depend mostly on non-commercial beekeepers to provide sufficient colonies for adequate pollination. Under optimal circumstances, beekeepers and fruit growers have mutual benefits from this cooperation as both honey and fruit yield increase. In those European countries with a monitoring scheme, acute bee poisoning incidents have decreased considerably and hardly cause problems at present. In recent years, some concerns arose around sublethal effects (i.e., behavioural changes) of chemical crop protection on bees, especially with regard to increased winter mortality. Even though short-term effects can indeed be induced in individually exposed bees, studies that exposed complete colonies did not reveal any long-term consequences at colony level. However, from the fruit growers' viewpoint, potential short-term effects on foraging behaviour are relevant as they can bear on pollination efficacy.

Development of a real-time PCR assay for measurement of yellow protein mRNA transcription in the desert locust Schistocerca gregaria: a basis for isolation of a peptidergic regulatory factor.

A major unresolved issue in insect endocrinology concerns the question of whether or not insects have sex hormones. Conclusive evidence in favor of the presence of such hormones awaits the establishment of appropriate bioassays in males. The cuticle of sexually mature males of the desert locust Schistocerca gregaria turns yellow in gregarious conditions only. Neither females nor isolated males ever turn yellow. The yellowing is due to the deposition in the cuticle of a male-specific Yellow Protein (YP), of which the amino acid sequence is known. In this paper, we describe the partial cloning of the cDNA encoding this Yellow Protein. The tissue distribution and temporal expression of the YP-mRNA is studied in detail using RT-PCR. Furthermore, an RT-PCR based bioassay was developed, which may serve as a reliable tool to help identify the hormones controlling the yellowing process. In addition to juvenile hormone, we have shown that a factor present in the brain-corpora cardiaca is involved in the yellow coloration, as injection of an extract induces the expression of YP-mRNA in isolated gregarious males.

Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies.

Insect cell cultures played central roles in unraveling many insect physiological and immunological processes. Regardless, despite imminent needs, insect cell lines were developed primarily from Dipteran and Lepidopteran orders, leaving many important insects such as Orthopteran locusts under-represented. Besides the lack of cell lines, the slow progress in development of in vitro techniques is attributed to poor communications between different laboratories regarding optimized primary cell cultures. Therefore, we report here about methods developed for primary cell culture of Locusta migratoria hemocyte and phagocytic tissue cells by which we could maintain viable hemocytes in vitro for over 5 d and phagocytic tissue cells for over 12 d. 2-Mercaptoethanol and phenyl-thiourea supplements in Grace's medium together with addition of fetal bovine serum 30 min after cell seeding resulted in a successful setup of the primary cell cultures and a week-long survival of the hemocytes and phagocytic tissue cells in vitro.

Characterization of the adipokinetic hormone receptor of the anautogenous flesh fly, Sarcophaga crassipalpis.

Adipokinetic hormone (AKH) is an insect neuropeptide mainly involved in fat body energy mobilization. In flies (Phormia regina, Sarcophaga crassipalpis), bugs (Pyrrhocoris apterus) and cockroaches (Periplaneta americana) AKH was also demonstrated to be involved in the regulation of digestion. This makes AKH an important peptide for anautogenous female flies that need to feed on a supplementary protein meal to initiate vitellogenesis, the large scale synthesis of yolk proteins and their uptake by the developing oocytes. Flesh fly AKH, originally identified as Phormia terraenovae hypertrehalosemic hormone (PhoteHrTH), functions through activation of the AKH receptor (AKHR). This is a G protein-coupled receptor that is the orthologue of the human gonadotropin-releasing hormone receptor. Pharmacological characterization indicated that the receptor can be activated by two related dipteran AKH ligands with an EC50 value in the low nanomolar range, whereas micromolar concentrations of the Tribolium castaneum AKH were needed. Consistent with the energy mobilizing function of AKH, the receptor transcript levels were most abundant in the fat body tissue. Nonetheless, Sarcophaga crassipalpis AKHR transcript levels were also high in the brain, the foregut and the hindgut. Interestingly, the receptor transcript numbers were reduced in almost all measured tissues after protein feeding. These changes may enforce the use of ingested energy carrying molecules prior to stored energy mobilization.

Structure, evolutionary conservation, and functions of angiotensin- and endothelin-converting enzymes.

Angiotensin-converting enzyme, a member of the M2 metalloprotease family, and endothelin-converting enzyme, a member of the M13 family, are key components in the regulation of blood pressure and electrolyte balance in mammals. From this point of view, they serve as important drug targets. Recently, the involvement of these enzymes in the development of Alzheimer's disease was discovered. The existence of homologs of these enzymes in invertebrates indicates that these enzyme systems are highly conserved during evolution. Most invertebrates lack a closed circulatory system, which excludes the need for blood pressure regulators. Therefore, these organisms represent excellent targets for gaining new insights and revealing additional physiological roles of these important enzymes. This chapter reviews the structural and functional aspects of ACE and ECE and will particularly focus on these enzyme homologues in invertebrates.

Insecticidal activity of the pyrimidine nucleoside analogue (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU).

The insecticidal activity of the antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was assessed in in vivo assays against the fall armyworm, Spodoptera frugiperda (JE Smith) (Lepidoptera, Noctuidae). BVDU, mixed into an artificial diet, caused a variety of effects, depending on the concentration used. Compared with controls, food intake was lower, larval growth was retarded and larval development was prolonged. The treated larvae formed smaller pupae and the hatching moths often showed morphogenetic defects. A higher mortality could be found in larval and pupal stages and was generally caused by moult disruption. A choice assay showed that BVDU has very slight feeding-deterrent properties, which only partly explain the toxic effects. The agent most probably acts through its cytostatic activity that has been described previously using cell lines of different insect species.

Locust cellular defense against infections: sites of pathogen clearance and hemocyte proliferation.

The locust cellular defense is mediated by hemocytes and hematopoietic tissue. In Locusta migratoria, the hemocytes and hematopoietic tissue mutually assist each other in clearing invading pathogens from circulation. A ß-1, 3-glucan infection induces nodule formation and apoptotic, TUNEL positive, cells in the hematopoietic tissue and massive loss of hemocytes in the circulation, calling for instant proliferation of hemocytes and hematopoietic tissue cells to assure continued host cellular defense. As the locust hematopoietic tissue persists at the adult stage, it was originally designated as being the major source for the replenishment process. Revisiting post infection hemocyte proliferation, using immunofluorescence based tests for DNA synthesis and mitosis, evidenced the lack of ß-1, 3-glucan induced cell proliferation in the hematopoietic tissue. Instead these tests identified the circulating hemocytes as the major source for hemocyte replenishment in the circulation. The hematopoietic tissue, however, undergoes a continuous, slow and infection independent regeneration, thereby accumulating potential phagocytes despite infection, and might serve a prophylactic role in containing pathogens in this swarming insect.

Identification and functional characterization of a novel locust peptide belonging to the family of insect growth blocking peptides.

Growth blocking peptides (GBPs) are recognized as insect cytokines that take part in multifaceted functions including immune system activation and growth retardation. The peptides induce hemocyte spreading in vitro, which is considered as the initial step in hemocyte activation against infection in many insect species. Therefore, in this study, we carried out a series of in vitro bioassay driven fractionations of Locusta migratoria hemolymph combined with mass spectrometry to identify locust hemocyte activation factors belonging to the family of insect GBPs. We identified the locust hemocyte spreading peptide (locust GBP) as a 28-mer peptide encoded at the C-terminus of a 64 amino acid long precursor polypeptide. As demonstrated by QRT-PCR, the gene encoding the locust GBP precursor (proGBP) was expressed in large quantities in diverse locust tissues including fat body, endocrine glands, central nervous system, reproductive tissues and flight muscles. In contrary, hemocytes, gut tissues and Malpighian tubules displayed little expression of the proGBP transcript. The bioactive peptide induces transient depletion of hemocytes in vivo and when injected in last instar nymphs it extends the larval growth phase and postpones adult molting. In addition, we identified a functional homologous hemocyte spreading peptide in Schistocerca gregaria.

Cloning and expression of the yolk protein of the tsetse fly Glossina morsitans morsitans.

Two major families of nutritional proteins exist in insects, namely the vitellogenins and the yolk proteins. While in other insects only vitellogenins are found, cyclorraphan flies only contain yolk proteins. Possible sites of yolk protein synthesis are the fat body and the follicle cells surrounding the oocyte. We report the cloning of the yolk protein of the tsetse fly Glossina morsitans morsitans, a species with adenotrophic viviparity. The tsetse fly yolk protein could be aligned with other dipteran yolk proteins and with some vertebrate lipases. In contrast to the situation in most fly species, only a single yolk protein gene was found in the tsetse fly. Northern blot analysis showed that only the ovarian follicle cells, and not the fat body represents the site of yolk protein synthesis.

Isolation and characterization of an angiotensin converting enzyme substrate from vitellogenic ovaries of Neobellieria bullata.

Vitellogenic ovaries of the gray fleshfly Neobellieria bullata contain a variety of unidentified substances that interact, either as a substrate or as an inhibitor, with angiotensin converting enzyme (ACE). We here report the isolation and characterization of the first ACE interactive compound hereof. This 1312.7 Da peptide with the sequence NKLKPSQWISL, is substrate to both insect and human ACE. It is a novel peptide that shows high sequence similarity to a sequence at the N-terminal part of dipteran yolk polypeptides (YPs). We propose to call it N. bullata ovary-derived ACE interactive factor or Neb-ODAIF. Both insect and human ACE hydrolyze Neb-ODAIF by sequentially cleaving off two C-terminal dipeptides. K(m) values of Neb-ODAIF and Neb-ODAIF(1-9) (NKLKPSQWI) for human somatic ACE (sACE) are 17 and 81 microM, respectively. Additionally, Neb-ODAIF(1-7) (NKLKPSQ) also interacts with sACE (K(m/i)=90 microM). These affinity-constants are in range with those of the physiological ACE substrates and suggest the importance of Neb-ODAIF and its cleavage products in the elucidation of the physiological role of insect ACE. Alternatively, they can serve as lead compounds in the development of new drugs against ACE-related diseases in humans.

Presence of angiotensin converting enzyme (ACE) interactive factors in ovaries of the grey fleshfly Neobellieria bullata.

Angiotensin converting enzyme (ACE) activity, defined as a captopril-inhibitable dipeptidyl carboxypeptidase activity towards 3H-hippurylglycylglycine, was demonstrated in haemolymph, testes and ovaries of the grey fleshfly Neobellieria bullata, hereby suggesting a physiological role for ACE in these particular tissues. While the ACE activity in haemolymph and testes reached relatively high levels, only minute ACE activity could be detected in ovaries throughout the entire vitellogenic cycle. Ovarian extracts of Neobellieria bullata do contain, however, in addition to Neb-TMOF, the Neobellieria bullata trypsin modulating oostatic factor which is an in vitro and a putative in vivo substrate of ACE in circulation, several other heat-stable molecules which individually function either as an ACE substrate or ACE inhibitor. Presumably these ACE interactive factors mask ACE activity in the fly ovaries, as measured by a classic substrate-binding assay. Purification and characterisation of these ACE substrates/inhibitors is in progress and is likely to facilitate the elucidation of the enigmatic physiological relevance of ACE in insects.

Neprilysins: an evolutionarily conserved family of metalloproteases that play important roles in reproduction in Drosophila.

Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model with which to analyze this protein family's function, we characterized the gene family in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi we individually targeted the five Nep genes most closely related to vertebrate neprilysin, Nep1-5, to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. Our results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates.

Lipophorins can adhere to dsRNA, bacteria and fungi present in the hemolymph of the desert locust: a role as general scavenger for pathogens in the open body cavity.

Desert locusts are characterized by a highly sensitive and effective RNA interference (RNAi) response. Moreover, delivery of dsRNA into the open body cavity will elicit potent silencing effects throughout the body. On the other hand, many other insect species, such as Bombyx mori and Drosophila melanogaster, lack the ability to efficiently spread the RNAi-signal. In this study, we demonstrated that, in the serum of the desert locust, lipophorins adhere to dsRNA-fragments. Lipophorins can be subdivided into high density and low density lipophorins (HDLp and LDLp), according to their buoyant density, and we showed that both types of lipophorins can interact with dsRNA fragments. Furthermore, in the presence of (gram-positive) bacteria or fungi, LDLp induce the formation of pathogen aggregates, while no clear aggregation effects were detected in the presence of HDLp.