RNA interference mortality points to noncircadian functions for the clock gene in the desert locust Schistocerca gregaria.

One of the core genes in the circadian regulation network is clock (clk). By forming a heterodimer with CYCLE (CYC) that binds on an E-box in the promoter region, it induces the transcription of other elements in the circadian transcriptional feedback loops and different clock output genes. In contrast to other insects, a clk double-stranded RNA (dsRNA) treatment is lethal in adults and fifth instar nymphs of the desert locust, Schistocerca gregaria, in a dose-dependent manner. Clk knock down fifth instar nymphs are able to undergo their imaginal moult but, depending on the amount of dsRNA, it takes them longer than the controls to reach adulthood. As adults, clk knock down animals do not develop their fat body and ovaries like the control animals. Therefore, we tested the expression of different genes involved in energy metabolism and reproduction to see the effect of the clk RNA interference knock down. Surprisingly, the expression of the vitellogenin gene was up-regulated in the clk knock down females who did not appear to invest their energy in egg development. Taken together, our results point out that the clk gene in the desert locust has an additional function in development besides its established role in maintaining the circadian rhythms in the brain.

Effects of sublethal doses of crop protection agents on honey bee (Apis mellifera) global colony vitality and its potential link with aberrant foraging activity.

Honey bees (Apis mellifera) are the most economically valuable pollinators of fruit crops worldwide. Taking into account bees' contributions to other flowering agricultural crops, about one-third of our total diet comes directly or indirectly from bee-pollinated plants. However, in recent years there increasingly have been worrisome alarm sounds on serious bee mortalities and mysterious disappearance of bees from beehives. Among several environmental factors (e.g. climate and bee pathogens), stress factors arising from agricultural practices can potentially play a role in bee losses. Detailed knowledge on the effects of plant protection products is essential to improve usage with minimal risks. In order to identify potential medium- and long-term effects, we followed up various sublethal contaminated hives during the prolongation of the fruit-growing season. More specifically, a large-scale experiment was conducted in which at four distinct locations (in the Limburg region of Belgium) four different bee colonies (representing three different contaminations -imidacloprid, fenoxycarb, indoxacarb- and a non-contaminated control hive) were thoroughly monitored every 2-7 days. Our observations point towards decays of overall colony vitality for several hives a couple of weeks after treatment, as indicated by a set of carefully assessed parameters including the total amount of active and dead bees, total surface of capped brood and overall colony weight. These outcomes could be linked to subtle differences in foraging activity between distinct hives. The implications of these results are discussed in terms of potential short-term and long-term consequences of disturbed foraging ability triggered by exaggerated exposure to sublethal doses of crop protection chemicals, and its potential impact on colony health.

Amfor expression in the honeybee brain: a trigger mechanism for nurse-forager transition.

The honeybee's colony fitness relies on an optimized age-dependent division of labor. Transition from nursing activities to foraging activities is associated with an increase in the expression of the Amfor gene. Ben-Shahar et al. [Ben-Shahar, Y., Robichon, A., Sokolowski, M.B., Robinson, G.E., 2002. Influence of gene action across different time scales on behavior. Science 296, 741-744] showed that the Amfor transcripts and their gene products are involved in regulating the transition from one task to the next. In this study, we investigated the trajectory of the expression of this gene in the brain over time. The expression pattern could contribute to our understanding of the involvement of Amfor in the transition process. Is there a gradual increase in transcript or a peak in expression triggering a downstream path of multiple differential gene expression? Hereto, bees were sampled from colonies containing marked 1-day-old bees every 2 or 3 days around the expected time of transition from nurse to forager, from day 13 to 25. To quantify Amfor transcript in the brain, we developed a real-time RT-PCR assay, based on Taqman technology, using fluorescent probes. Results revealed a trigger mechanism rather than a continued elevation of Amfor expression. The appearance of an Amfor expression peak suggests that under normal physiological conditions foraging behavior is, at least in part, due to a trigger-effect of Amfor.

Cyclorraphan yolk proteins and lepidopteran minor yolk proteins originate from two unrelated lipase families.

Vitellogenins, cyclorraphan yolk proteins and lepidopteran minor yolk proteins are three classes of female-specific proteins that serve as an embryonic nutritional store. Similarity to vertebrate lipid-binding proteins was established for vitellogenins and yolk proteins, vitellogenins being related to apolipoprotein B and yolk proteins to lipases. Recently, similarity between yolk proteins and minor yolk proteins was reported and it was suggested that yolk proteins are more related to minor yolk proteins than to vertebrate lipases. In this study, we cloned five additional yolk proteins from the grey fleshfly Neobellieria bullata, formerly known as Sarcophaga bullata. We used this sequence data, combined with sequence data retrieved from the NCBI protein database to evaluate the yolk protein-lipase and the yolk protein-minor yolk protein relationship. We found no similarity between yolk proteins and minor yolk proteins, but we showed that yolk proteins are related to a family of lipases containing vertebrate hepatic and pancreatic lipases while minor yolk proteins are related to a family of lipases containing vertebrate gastric and lingual lipases. The fact that three different classes of yolk storage proteins show similarity to three different classes of vertebrate lipid-binding proteins strongly suggests that this lipid-binding feature is important for insect yolk storage proteins.

Molecular evidence for the expression of angiotensin converting enzyme in hemocytes of Locusta migratoria: stimulation by bacterial lipopolysaccharide challenge.

The presence of angiotensin converting enzyme (ACE) in insects has been reported many times, but numerous questions about the functional role of this enzyme in insects remain. Here we show by RT-PCR experiments that ACE has a wide tissue distribution in Locusta migratoria, suggesting diverse roles for this enzyme in the locust. Immune challenge through injection of bacterial lipopolysaccharides resulted in a tenfold increase of ACE gene transcripts in the hemocytes and is suggestive for a role of ACE in the cellular defense of the locust. However, phenotypic knockout experiments with the ACE inhibitor captopril showed that ACE is not essential for the efficient clearance of injected E. coli bacteria.

Search for phase specific genes in the brain of desert locust, Schistocerca gregaria (Orthoptera: Acrididae) by differential display polymerase chain reaction.

Differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) in combination with semi-quantitative RT-PCR was used to compare differences in gene expression between the solitary and gregarious phase of Schistocerca gregaria. Twenty-six primer combinations were used, which produced 8 differential bands. Two out of the 8 differentials, one typical for the solitary and one for the gregarious phase, were further analyzed by semi-quantitative RT-PCR. The expression level of the solitary phase specific gene (SSG) was 2 times higher in solitary animals as compared to gregarious ones, while the gregarious specific gene (GSG) gave a 4-fold higher expression level in gregarious animals than in solitaries. Sequence analysis demonstrated that SSG does not belong to a known gene family, while the GSG belongs to the SPARC protein family.

An endothelin-converting enzyme homologue in the locust, Locusta migratoria: functional activity, molecular cloning and tissue distribution.

Endothelin-converting enzyme is the key enzyme in the process of endothelin production. Endothelin is a peptide that plays an important role in vasoconstriction and the development of neural crest-derived cells in vertebrates. Activity assays performed on membrane extracts from Locusta migratoria brain revealed the existence of a protease activity responsible for the formation of mature endothelin-1 from its precursor, big endothelin. Cloning experiments led to a cDNA sequence (Lom ECE) with an open reading frame of 727 amino acid residues displaying all the characteristic ECE features. A comparison of ECE activity levels among different tissues of the locust showed a high enzyme activity in the gonads and midgut. RT-PCR experiments showed a wide tissue distribution of Lom ECE mRNA, with transcription being most abundant in brain tissue.

Captopril, a specific inhibitor of angiotensin converting enzyme, enhances both trypsin and vitellogenin titers in the grey fleshfly Neobellieria bullata.

A strong and constitutive angiotensin converting enzyme- or ACE-like activity was demonstrated in the hemolymph of the adult grey fleshfly Neobellieria bullata. In a competition assay, the N. bullata trypsin modulating oostatic factor (Neb-TMOF) was confirmed to be an in vitro substrate for this circulating Neb-ACE. Oral uptake of captopril, a selective and specific inhibitor of ACE, resulted in a complete phenotypic knockout of circulating ACE activity. When compared with control animals, captopril-fed female flies showed an increase in the liver meal-induced trypsin peak in the midgut and elevated levels of protein meal-induced yolk polypeptides in the hemolymph. The latter effect was not due to a slower vitellogenin uptake by the ovaries, because oocyte growth was not affected by the captopril treatment. The apparent synergism between the demonstrated ACE functionality and the previously reported effects of the oostatic peptide Neb-TMOF are discussed in the context of our recent finding that Neb-TMOF represents a prime candidate for being the first known in vivo substrate for circulating insect ACE. Arch.

In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata.

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.

Proteolytic breakdown of the Neb-trypsin modulating oostatic factor (Neb-TMOF) in the hemolymph of different insects and its gut epithelial transport.

The degradation of the unblocked hexapeptide, trypsin modulating oostatic factor of the flesh fly Neobellieria (Sarcophaga) bullata (Neb-TMOF) was studied in vitro in the hemolymph of the lepidopteran Spodoptera frugiperda, the orthopteran Schistocerca gregaria and the dictyopteran Leucophaea maderae. The half-life in the different species varied from approximately 3min in L. maderae to approximately 25min in S. gregaria. Purification of the degradation products and ESI-Qq-oa-Tof mass spectrometry revealed the fragments Asn-Pro-Thr-Asn, Leu-His and Asn-Pro, which were the same in the hemolymph of all species. Except in Leucophaea, Neb-TMOF was cleaved in dipeptides starting from the C-terminus and the reaction could be, at least partially, inhibited by captopril. These observations suggest that a dipeptidase, which has very similar enzymatic properties as mammalian angiotensin converting enzyme (ACE) and which circulates in the hemolymph, apparently is involved in the breakdown of Neb-TMOF and might be a common but not a universal enzyme in insect hemolymph.The introduction of Neb-TMOF into the gut of S. gregaria with the help of a capillary tube (intubation) demonstrated that the intact peptide is able to cross the gut epithelium and to appear in the hemolymph compartment. Since [3H]-inulin, which is too large to cross cell membranes, was found to penetrate the gut walls at a measurable rate, the paracellular pathway might be also permeable to smaller peptides. There was indeed a clear correlation between the molecular weight of inulin, Neb-TMOF, and inositol and the rate of penetration of these compounds through the gut epithelium to the hemolymph. These are promising findings in view of a potential use of such peptides for insect control purposes.

The multifunctional deoxynucleoside kinase of insect cells is a target for the development of new insecticides.

The antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was found to be an efficient substrate for recombinant Drosophila melanogaster-deoxyribonucleoside kinase with a K(m) of 4.5 microM and a V(max) of 400 nmol/microg protein/h compared with 1.3 microM and 62.5 nmol/microg protein/h, respectively, for the natural substrate thymidine. Mammalian cytosolic thymidine kinase-1 does not recognize BVDU as a substrate. In sharp contrast to mammalian cells, the insect D. melanogaster and Spodoptera frugiperda (Sf) embryonic cells proved highly sensitive to the cytostatic action of BVDU. BVDU was efficiently metabolized to its 5'-mono-, 5'-di- and 5'-triphosphate derivatives in the insect cell cultures and abundantly incorporated into the insect cell DNA. BVDU prevented the D. melanogaster cells to initiate the S phase of their cell cycle, and exposure of S. frugiperda cells to BVDU led to a dose-dependent retardation of the insect cells in the S phase of their cell cycle. Both inhibition of nucleic acid synthesis (through the 5'-triphosphate of BVDU) and inhibition of thymidylate synthase (through the 5'-monophosphate of BVDU) would account for the cytostatic activity of BVDU against the insect cells. Because of the virtual lack of cytotoxicity of BVDU against mammalian cells, the drug should be considered highly selective in its cytostatic action against the insect cells. When added to the food of S. frugiperda larvae, BVDU caused a remarkable decrease in the weight gain of the larvae and heavily compromised the transformation of the larvae to the pupae and their subsequent adult (moth) phase. Our data indicate that insect multifunctional deoxyribonucleoside kinase should be considered an entirely novel and attractive target in the development of new nucleoside types of highly selective insecticidal drugs.

Reproduction and love: strategies of the organism's cellular defense system?

A novel view is presented which states that primordial germ cells and their descendants can be regarded as 'cancerous cells' which emit signals that activate a whole array of cellular defensive mechanisms by the somatoplasm. These cells have become unrestrained in response to the lack of typical cell adhesion properties of epithelial cells. From this point of view: (1) the encapsulation of oocytes by follicle cells, vitelline membrane and egg shell; (2) the suppression of gonadal development in larval life; (3) the production of sex steroid hormones and of vitellogenin; and (4) the expulsion of the gametes from the body fit into a general framework for a defense strategy of the somatoplasm against germ line cells. Accordingly, the origin of sexual reproduction appears to be a story of failure and intercellular hostility rather than a 'romantic' and altruistic event. Yet, it has resulted in evolutionary success for the system in which it has evolved; probably through realizing feelings of 'pleasure' associated with reproduction.

"Insects do not have sex hormones": a myth?

Mammals have two genes (SRY and DMT1) for testis formation-androgenesis, an anti-testis gene, DAX1, an anti-Müllerian duct hormone, and steroid sex hormones. Drosophila uses the sex-lethal, transformer, and doublesex genes for sexual differentation and is supposed to lack sex hormones. However, the statement that insects do not have sex hormones loses much of its credibility if one considers (1) the classical endocrinological work on sexual differentiation in the firefly Lampyris and in the hevea tussock moth Orgyia; (2) the recent identification of an androgenic hormone and its role in sex determination in the isopod Armadillidium; (3) the similarity between steroidogenic factor 1 (SF-1) of mammals and fushi tarazu factor 1 (FTZ-F1) of Drosophila; and (4) the steroidogenic effect of gonadotropins secreted by the brain of female locusts and mosquitoes and of male gypsy moth. In our model, based on data from the literature, ecdysone, when present in high concentrations, might function as an androgenic sex steroid. It is also the precursor of 20-OH-ecdysone, which is the moulting hormone of insects, and in vitellogenic females of many species, the counterpart of estrogens as well. Other gender-specific hormones are likely to exist in the brain-gonad axis.

Widespread occurrence of the micro-organism Wolbachia in ants.

For more than 20 years, sex allocation in hymenopteran societies has been a major topic in insect sociobiology. A recurring idea was that relatedness asymmetrics arising from their haplodiploid sex determination system would lead to various parent-offspring conflicts over optimal reproduction. A possible weakness of existing theory is that only interests of nuclear genes are properly accounted for. Yet, a diversity of maternally transmitted elements manipulate the reproduction of their host in many solitary arthropod groups. The bacterium Wolbachia is a striking example of such a selfish cytoplasmic element, with effects ranging from reproductive incompatibility between host strains, induction of parthenogenesis and feminization of males. This paper reports on a first PCR-based Wolbachia screening in ants. Out of 50 Indo-Australian species, 50% screened positive for an A-group strain. One of these species also harboured a B-group strain in a double infection. Various factors that might explain the unusually high incidence of Wolbachia in ants are discussed. In general, Wolbachia may represent a widespread and previously unrecognized party active in the conflicts of interest within social insect colonies.

Immunocytochemical distribution of angiotensin I-converting enzyme-like immunoreactivity in the brain and testis of insects.

Angiotensin converting enzyme (ACE) is Zn2+ metallopeptidase which plays an important role in blood pressure homeostasis in mammals and other vertebrates. Homologues of ACE involved in the biosynthesis of mammalian peptide hormones have also been identified in the insects, Musca domestica, Drosophila melanogaster and Haematobia irritans exigua. In the pursuit of the biological role of insect ACE, this work focused on the tissue and cellular distribution of ACE in several insect species. The localisation of ACE in the central nervous system and reproductive tissues from a number of insect species suggests that ACE is of physiological importance in these tissues. By means of an antiserum to housefly ACE, we found that ACE-like immunoreactivity was abundantly present in the neuropil areas of the brain of all insects investigated, suggesting a role for ACE in the metabolic inactivation of peptide neurotransmitters. Especially in the fleshfly, Neobellieria bullata neuropile staining is abundant. In the cockroach Leucophaea maderae, immunoreactive staining was abundant in the neuronal perikarya as well as in the neuropilar regions. Staining in neurosecretory cells was also observed in the brains of the lepidopteran species, Bombyx mori and Mamestra brassica. The localisation of ACE in neurosecretory cells is consistent with the role as a processing hormone, involved in the generation of active peptide hormones. ACE was found to be co-localised with peptides of the FXPRLamide family in M. brassica and in B. mori, suggesting a role for the biosynthesis of these hormones. Finally, we found ACE-like immunoreactivity in the testis of Locusta migratoria, N. bullata and Leptinotarsa decemlineata, providing additional evidence for its important role in insect reproduction.

Molecular sequencing and modeling of Neobellieria bullata trypsin. Evidence for translational control by Neobellieria trypsin-modulating oostatic factor.

Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reversed transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and sequenced. The 3' end of the gene (300 bp) was amplified by means of the rapid-amplification-of-cDNA-ends method, cloned and sequenced. The deduced protein sequence of 254 amino acids exhibited 46% identity to Drosophila trypsin and 32% identity to Anophiline trypsin and Aedes trypsin. Three-dimensional models of Neobellieria trypsin and Drosophilia trypsin were built and compared. Both models contain two domains of beta-barrel sheets as was shown by means of X-ray crystallography of mammalian trypsin. The catalytic active site is composed of the canonical triad of His42, Asp87 and Ser182 whereas Asp176 sits as the bottom of the specificity pocket. Southern blot analysis suggested that Neobellieria trypsin is encoded by one gene. Northern blot analysis showed that an early trypsin transcript is found in the midgut of sugar-fed females. This message disappeared after a liver meal, and was replaced by a late transcript. Injection of trypsin-modulating oostatic factor (TMOF) at 10(-9) M prevented the disappearance and the translation of the early transcript. TMOF did not prevent the appearance of the late transcript. However, in the presence of the hormone the late transcript was not translated. Thus, TMOF is the biological signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut.

Characterization of a cloned locust tyramine receptor cDNA by functional expression in permanently transformed Drosophila S2 cells.

The cDNA for Tyr-Loc, a G protein-coupled receptor that clearly shows homology to a number of mammalian and fruit fly receptors for biogenic amines, was cloned from the nervous system of Locusta migratoria. Functional expression of the cloned cDNA was obtained in cultured insect cells, i.e., in Spodoptera SF9 cells using a baculoviral expression system and in stably transformed Drosophila Schneider 2 (S2) cells. Multiple copies of the receptor expression construct are inserted into the genome of these permanently transformed cells. The expression of the receptor cDNA was driven by the upstream sequences of a Bombyx mori baculoviral immediate early gene. Tyramine shows a much higher binding affinity to this receptor than other possible endogenous ligands. It also reduces forskolin-induced cyclic AMP production in the permanently transformed S2 cells. The pharmacological profile of the Tyr-Loc receptor is distinct from that of any locust receptor-type described so far, but it is similar to that of the Drosophila tyramine/octopamine receptor. In the locust CNS, the Tyr-Loc mRNA is not present in the distal part of the optic lobes but has a widespread distribution in the brain and the ventral nerve cord.

Folliculostatins, gonadotropins and a model for control of growth in the grey fleshfly, Neobellieria (sarcophaga) bullata.

The sequences of two folliculostatic peptides of the fleshfly Neobellieria bullata have been determined recently. The first peptide (Neb-TMOF: H-NPTNLH-OH), originates from a 75 kDa precursor protein found in vitellogenic oocytes. The hexapeptide directly inhibits the synthesis of trypsin-like enzymes in the gut, and thus lowers the concentration of yolk polypeptides in the hemolymph. It also inhibits the biosynthesis of ecdysone in the larval ring gland. Therefore, it could also be named prothoracicostatic hormone (Neb-PTSH). The second peptide (Neb-colloostatin: H-SIV-PLGLPVPIGPIVVGPR-OH) acts on previtellogenic follicles and is a cleaved product of a collagen-like precursor molecule. Our results indicate that peptides that are cleaved from matrix proteins could act as growth-inhibiting factors. Gonadotropin releasing hormone (GnRH)-immunolike peptides were not identified, but progress is being made in the isolation and characterization of factors which stimulate cAMP production by the ovary. Using these results, a novel model of growth control in which matrix proteins play an important role as a potential source of growth regulators has been developed.

Neb-colloostatin, a second folliculostatin of the grey fleshfly, Neobellieria bullata.

During the purification of trypsin-modulating oostatic factor (TMOF) of the grey fleshfly Neobellieria bullata, a new factor with oostatic activity was discovered. We report herein its purification, primary structure and effects on oocyte development. Its amino acid sequence was determined as H-SIVPLGLPVPIGPIVVGPR-OH. Due to structural sequence similarities with parts of several known collagens and its oostatic activity, we named it Neb-colloostatin. The synthetic peptide inhibits yolk uptake by previtellogenic oocytes and might have a role in the absence of yolk deposition in penultimate oocytes. Neb-colloostatin does not inhibit trypsin biosynthesis in the gut or ecdysone biosynthesis by larval ring glands. It decreases vitellogenin concentrations in the hemolymph by an unknown mode of action. The role of extracellular matrix proteins in the feedback control of growth is discussed.

Glycoform heterogeneity of porcine interferon-gamma expressed in Sf9 cells.

Porcine interferon-gamma (SfPoIFN-gamma) was expressed with high efficiency in Spodoptera frugiperda (Sf9) cells by means of the baculovirus expression system. Up to 10(5) U/ml of antivirally active SfPoIFN-gamma could be tracked down in the culture medium at 64 h postinfection. Three proteins (17, 19, and 21 kDa), which under nondenaturing conditions primarily exist as mutual-dimeric combinations, were purified by immunoaffinity chromatography. Carbohydrate labeling and kinetic deglycosylation studies suggested that the 19- and 21-kDa proteins are N-glycosylated variants of a single 17-kDa protein carrying no N-linked sugars, in which one respectively two N-glycosylation sequons are occupied by glycans of 2 kDa. Both the quantitative recovery of SfPoIFN-gamma from a Con A column at 0.2 M methyl-alpha-mannopyranoside and the results of lectin blots, revealing strong affinity of the 19- and 21-kDa species for Galanthus nivalis agglutinin, support the presence of N-glycosidically linked high mannose-type chains in the carbohydrate moiety of SfPoIFN-gamma. Intriguingly, both 19- and 21-kDa glycoforms, but not their sialidase-treated derivatives, showed clear reactivity with the Sambucus nigra and Maackia amurensis agglutinins. These agglutinins specifically recognize sialic acid linked alpha(2-6) and alpha(2-3), respectively, to penultimate galactose residues. Their affinity for the larger glycoforms of PoIFN-gamma suggests that the biosynthetic pathways in Sf9 cells are able to modify oligomannose structures to complex or hybrid glycans.