Sequencing and biological effects of an adipokinetic/hypertrehalosemic peptide in the stick insect, Baculum extradentatum

The corpora cardiaca of the Vietnamese stick insect, Baculum extradentatum, contain a member of the adipokinetic hormone/red pigment-concentrating hormone/hypertrehalosemic hormone (AKH/RPCH/HrTH) family of peptides whose sequence is identical to that originally described for the Indian stick insect, Carausius morosus. This decapeptide, Carmo-HrTH-II (pELTFTPNWGTa), has both hypertrehalosemic and cardioacceleratory activity in B. extradentatum, and hyperlipaemic activity in locusts. Reversed-phase high performance liquid chromatography (RP-HPLC) of corpora cardiaca extract followed by MALDI-TOF MS/MS also revealed a novel modification of a second peptide in B. extradentatum: the tryptophan residue at position 8 is post-translationally modified to kynurenine.     

Juvenile hormone and methyl farnesoate production in cockroach embryos in relation to dorsal closure and the reproductive modes of different species of cockroaches

Juvenile hormone (JH), produced by the corpora allata (CA), is first detectable after dorsal closure, a conspicuous event in embryogenesis. The present research found that the timing of dorsal closure was consistently at about 45% of the total embryonic development time across most of the oviparous and ovoviviparous cockroach species examined. These included the ovoviviparous cockroaches Blaberus discoidalis, Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, Phoetalia pallida, Schultesia lampyridiformis, and Panchlora nivea, as well as the oviparous cockroaches Blatta orientalis, Periplaneta americana, Eurycotis floridana, and Supella longipalpa. However, the only known viviparous cockroach Diploptera punctata completed dorsal closure at 20.8% of embryo development time. Methyl farnesoate (MF), the immediate precursor of JH III, is considered a functional molecule in crustaceans; however, in insects its function is still unclear. To understand the role of JH and MF in cockroach embryos, I compared JH and MF biosynthesis and release in several cockroach species of known phylogenetic relationships. Using a radiochemical assay, the present research showed that cockroach embryos representing all three reproductive modes produced and released both JH and MF, as previously shown for B. germanica, N. cinerea, and D. punctata. Members of a pair of embryonic CA from B. discoidalis, B. fumigata, R. maderae, and D. punctata were incubated with and without farnesol. MF accumulated in large amounts only in CA of R. maderae in the presence of farnesol, which indicates that control of the last step of biosynthesis of JH, conversion of MF into JH by MF epoxidase, is probably a rate-limiting step in this species.     

Vitellogenin of the cockroach,Leucophaea maderae: nucleotide sequence,structure and analysis of processing in the fat body and oocytes

A cDNA encoding vitellogenin (Vg) of the cockroach, Leucophaea maderae was cloned and sequenced. The deduced amino acid sequence consisting of 1913 residues (including 15 residues for a putative signal peptide) was obtained. Amino-terminal sequence analysis demonstrated that the pro-Vg was cleaved into four polypeptide 'subunits' following the three consensus RXXR cleavage site sequences, which were secreted as four Vg polypeptides (apparent molecular weights = 112-, 100-, 92- and 55-kD), sequestered, and deposited in the egg as four respective vitellin (Vn) polypeptides. There was, however, an additional 90-kD Vn polypeptide existed in the egg. We show that this polypeptide is a processed product from 92-kD Vn polypeptide. Northern blot analysis of poly (A)+ RNA reveals that mRNA coding for Vg is present only in the female fat body cells but neither in the ovary nor in the male fat body cells. The deduced amino acid sequence contained a serine-rich stretch at the C-terminal region. This stretch occurred also in Vgs of Periplaneta americana (Vg1 and Vg2) and Blattella germanica. The Vg of L. maderae had 26% and 31% homology with those of P. americana (Vg1 and Vg2) and B. germanica, respectively. Phylogenetic analysis (neighbour-joining) was made using four cockroach Vgs and the tree was compared with other molecular and conventional phylogenetic trees.     

The molecular evolution of the allatostatin precursor in cockroaches

Allatostatins (ASTs) of the Tyr/Phe-Xaa-Phe-Gly Leu/Ile-NH2 family are a group of insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have obtained genomic DNA sequences that specify the preproallatostatin precursor for the cockroaches, Blatta orientalis, Blattella germanica, Blaberus (cranufer and Supella longipalpa. The sequences obtained are similar to those of Diploptera punctata and Periplaneta americana reported previously. The precursors of all these cockroach species are similar in size, and the organization of the ASTs that they contain (there are 13 or 14, depending on the species) have been conserved. With the sequences of these precursors, and using the homologous sequence in the orthopteran Schistocera gregari as an outgroup, a phylogenetic analysis using parsimony was carried out. The dendrograms obtained from these analyses. using the amino acid as well as the nucleotide sequences, are comparable with current models for cockroach phylogeny. Parsimony analysis was also used to study the genealogy of the different ASTs within the same precursor. Results suggest that the AST sequences were generated through a process of internal gene duplication which occurred before these species diverged from each other in evolutionary time.     

昆虫激肽的生理功能及机制

昆虫激肽是一类高度保守的小分子神经活性物质,自其从马德拉蟑螂脑中分离得到至今,人们在多种无脊椎动物体内均发现了这一激肽家族成员。它们具有促进昆虫后肠收缩、马氏管扭动、原尿分泌,调节血淋巴量和水盐平衡,使马氏管跨膜电位去极化,抑制昆虫体内消化酶释放、幼虫体重增长等功能。然而,天然的昆虫激肽很容易被蛋白酶所降解,因此须对其进行结构改造及构效关系研究,以开发出更有潜力的假肽和非肽模拟抗酶解昆虫激肽类似物,对今后实现环境友好型害虫防治策略具有重要意义。     

Orcokinins in insects and other invertebrates

Orcokinin (NFDEIDRSGFGFN) and orcokinin homologues are crustacean peptides eliciting potent myotropic effects in gut tissues. Through HPLC purification of brain extract of the cockroach Blattella germanica, we isolated the first insect orcokinin (NFDEIDRSGFNS). This insect orcokinin-like peptide do not show myotropic properties in B. germanica gut tissues. Gene database search using orcokinin precursor sequences of the crustacean Procambarus clarkii led to putative homologues found in non-crustacean groups, including the mosquito Anopheles gambiae and the nematode Caenorhabditis elegans.     

Distribution of Oriental and German cockroaches, Blatta orientalis and Blattella germanica (Dictyoptera), in the United Kingdom

Results are presented of a survey carried out by Rentokil Ltd on the distribution of the Oriental cockroach Blatta orientalis L. and the German cockroach Blattella germanica L. in the United Kingdom. The known ranges of both species are increased considerably by the findings of the survey, with several new vice-county records for Scotland (including the Western Isles), England and Wales. The frequency at which Blatta orientalis was observed in outdoor habitats may indicate that this species sometimes spreads to new areas without human assistance.     

Silencing allatostatin expression using double-stranded RNA targeted to preproallatostatin mRNA in the German cockroach

YXFGL-NH(2) family allatostatins (ASTs) were isolated from cockroach brain extracts based on their capacity to inhibit juvenile hormone (JH) biosynthesis in corpora allata (CA) incubated in vitro. Subsequently, the inhibitory activity of synthetic ASTs was demonstrated experimentally, although these peptides were shown to be active as JH inhibitors only in cockroaches, crickets, and termites. Here, we sought to examine whether ASTs are true physiological regulators of JH synthesis. To this end, we used RNA interference methodologies and the cockroach Blattella germanica as a model. Treatments with double-stranded RNA targeting the allatostatin gene in females of B. germanica produced a rapid and long-lasting reduction in mRNA and peptide levels in both brain and midgut during the reproductive cycle. Nevertheless, while brain AST levels were reduced approximately 70-80%, JH synthesis did not increase in any of the age groups tested.     

Reproductive effects in German cockroaches by ecdysteroid agonist RH-0345, juvenile hormone analogue methoprene and carbamate benfuracarb

Blatta germanica is the more prevalent cockroach species in Algeria. In the present study, we tested the effect on reproduction in B. germanica of two insect growth regulators, RH-0345, a benzoylhydrazine analogue that mimics the action of 20-hydroxyecdysone, and methoprene, one of the most commercially important juvenile hormone analogues, and a novel carbamate insecticide, benfuracarb. The compounds were applied topically (10 and 20 microg/insect for RH-0345, and 1 and 10 microg/insect for methoprene) or orally administrated (at 2% for benfuracarb) on newly emerged females and evaluated on reproductive events during the adult life (2, 4 and 6 days). Treatment with RH-0345 and benfuracarb reduced significantly the number of oocytes, the size and the volume of the basal oocyte during the experimental period. Methoprene distorted the ovarian development since it caused a significant reduction in the number of oocytes at 2, 4 and 6 days for the two tested doses, and an increase in oocyte size at 2, 4 and 6 days with 1 microg and a decrease with 10 microg. In a second series of experiments, the effects of these compounds were assayed on the ovarian proteins. Data from biochemical analysis revealed that RH-0345 and benfuracarb reduced the ovarian amounts of proteins, while treatment with methoprene increased it during the sexual maturation.     

Vitellogenin of Blattella germanica (L.) (Dictyoptera, blattellidae): nucleotide sequence of the cDNA and analysis of the protein primary structure

The cloning and sequencing of a cDNA of the vitellogenin gene from the cockroach Blattella germanica is reported. It is 5,749 nucleotides long and encodes an amino acid sequence of 1,862 residues (including a putative signal peptide of 17 residues). The vitellogenin sequence includes a long serine-rich stretch between amino acids 322 and 349, and two other stretches between amino acids 1691 and 1740. The vitellogenin of B. germanica shows a notable similarity (between 32 and 42%) to those described in other insects, and its alignment shows a high number of motifs conserved in all species, especially in the subdomains I-V. Non-parsimony methods (Neighbor Joining) of phylogenetic analysis of the insect vitellogenin sequences gave a tree showing a topology that is, in general, congruent with the currently accepted insect phylogenetic schemes. Arch.     

Studies on the cellular defense reactions of the Madeira cockroach, Leucophaea maderae: nodule formation in response to injected bacteria

Nodules were formed in the Madeira cockroach, Leucophaea maderae, in response to injections of low doses (3 x 10(4) bacteria/insect) of three strains of Bacillus cereus and Escherichia coli K12 D31. The most pathogenic strain of bacteria used, B. cereus B1, produced the greatest cellular response, while the least pathogenic, E. coli K12 D31, injected at the same dose, caused little nodule formation. Similarly, nodules were generally found to be larger following injection of pathogenic bacteria such as B. cereus B1 than to the weak pathogen, E. coli K12 D31. There was, however, no difference in the extent of nodule formation with the four bacterial strains/species if they were heat killed prior to injection. Histologically, the nodules formed in response to all bacterial species employed were similar, with a central necrotic core enclosing cell debris and occasional bacteria, and an outer, thin sheath of plasmatocyte-like hemocytes. Possible reasons for the enhanced cellular reactivity observed in L. maderae to pathogenic bacteria are discussed.     

Identification of multiple peptides homologous to cockroach and cricket allatostatins in the stick insect Carausius morosus

Eighteen peptides were isolated from brain extracts of the stick insect Carausius morosus. The peptides were purified in four steps by high-performance liquid chromatography, monitored by their ability to inhibit juvenile hormone biosynthesis by corpora allata of the cricket Gryllus bimaculatus in vitro, and chemically characterised by Edman degradation and mass spectrometry. We obtained complete primary-structure information for nine peptides, four of which belong to the peptide family characterised by a common C-terminal pentapeptide sequence -YXFGLamide. The remaining five belong to the W(2)W(9)amide peptide family, nonapeptides characterised by having the amino acid tryptophan in positions 2 and 9. The amino-acid sequence of two other peptides could not be completely resolved by means of Edman degradation; however, these peptides could be allocated to the -YXFGLamide and the W(2)W(9)amide family, respectively, by comparison of retention times, co-elution and mass spectrometry. Both classes of neuropeptides strongly inhibit juvenile hormone biosynthesis in crickets but show no inhibiting effect on the corpora allata of the stick insect.     

Isolation of the hypertrehalosaemic factors I and II from the corpus cardiacum of the Indian stick insect, Carausius morosus, by reversed-phase high-performance liquid chromatography, and amino-acid composition of factor II

The separation of two peptides with hypertrehalosaemic activity from the corpus cardiacum of the Indian stick insect, Carausius morosus, was achieved by reversed-phase high-performance liquid chromatography using a Nucleosil C-18 column with a trifluoroacetic acid/acetonitrile gradient. The eluant was monitored at 210 nm and the hypertrehalosaemic activity was detected in ligated stick insects using a bioassay. In addition, the isolated material was potent in causing hyperlipaemia in migratory locusts and also in raising blood carbohydrates in the American cockroach. The amino-acid composition of the major peptide, hypertrehalosaemic factor II, was determined after acid hydrolysis with HCl or methanesulfonic acid. The analyses suggest that factor II is a nonapeptide which contains the following amino-acid residues: Asp, 2 Thr, Glu, Pro, Gly, Leu, Phe and Trp. This composition is almost identical to that of locust adipokinetic hormone I, lacking only one Asp residue.     

Innervation of the foregut of the cockroach Leucophaea maderae and inhibition of spontaneous contractile activity by callatostatin neuropeptides

Abstract. The innervation of the gut of the cockroach Leucophaea madera (F.) has been studied by means of wholemount immunocytochemistry with antisera raised against Leu-callatostatin, a cockroach allatostatin homologue identified from neuropeptide isolation and gene studies in the blowfly Calliphora vomitoria. Leu-callatostatin-imunoreactive neurones in the brain, with axon trajectories in the stomatogastric nervous system, innervate the foregut and midgut. Neurones in the last abdominal ganglion supply the hindgut and the midgut via the proctodeal nerve. In addition to a rich callatostatin-immunoreactive nerve supply, the midgut, including the midgut caeca, contain numerous callatostatin-immunoreactive endocrine cells. Physiological studies show that the spontaneous contractile activities of the foregut, but not the hindgut, are inhibited by callatostatin neuropeptides. Leu-callatostatin 3 was the most potent of the range of Leu-and Met-callatostatins tested, with a dose-dependent response between 10^-13 and 10^-7 M. This is similar to the results obtained with the previously identified myoinhibitory peptide of L. maderae , leucomyosuppressin. However, this peptide, with a different type of structure to the allatostatins, inhibits both foregut and hindgut motility equally. Experiments with a series of analogues of the Met-callatostatins showed that the free acid (as opposed to the carboxyamidated peptide) and N-terminally truncated peptides were inactive. These morphological and physiological results are thought to be representative of the, as yet unidentified, naturally occurring allatostatin homologues of L. maderae. This family of peptides should be added to the increasing list of insect gut myoinhibitory substances.     

Cockroaches as vectors of Sarcocystis muris and of other coccidia in the laboratory.

To assess the roles of the German cockroach (Blatella germanica) and the American cockroach (Periplaneta americana) in the transmission of Sarcocystis muris and of 3 other coccidia of cats-Toxoplasma gondii, Isospora felis, and Isospora rivolta, cockroaches exposed to feces containing these coccidia were periodically fed to mice, as was a portion of the fecal matter. Sarcocystis muris sporocysts, which in feces remained infectious for at least 20 days, were also transmitted to mice by P. americana for at least 20 days and by B. germanica for 5 days after exposure to infectious feces. Toxoplasma gondii oocysts were transmitted by P. americana intermittently up to 10 days, but by B. germanica only immediately after exposure to feces. Oocysts of 2 species of Isospora, when associated with fecal matter, remained infectious for 20 days. Those of I. rivolta were transmitted by both cockroach species for 10 days, but I. felis was transmitted only by by B. germanica, and for only 2 days.     

Neuronal co-localization of different isoforms of tachykinin-related peptides (LemTRPs) in the cockroach brain

Seven isoforms of tachykinin-related peptides (TRPs) have been isolated from the brain of the cockroach Leucophaea maderae. These peptides (LemTRP-1, 2, and 5-9) share the C-terminal sequence GFX(1)GX(2)Ramide (where X(1) and X(2) are variable residues). In order to determine the neuronal distribution of several of these LemTRP isoforms, we raised antisera to their variable N-termini. Antisera to LemTRP-1, 2, 3, 7, and 8 were utilized for immunocytochemistry on cryostat sections of the L. maderae brain. As expected, the gut peptide LemTRP-3 was not detected in the brain, and the antisera to LemTRP-1, 2, and 7 labeled the same sets of neurons in different regions of the brain. These neurons could also be labeled with antisera raised to the more conserved C-termini of LemTRP-1 and the locust TRP LomTK-I. The antiserum to LemTRP-8 predominantly labeled a set of neurons distinct from that seen with any other N- or C-terminus-directed antisera, suggesting that it recognizes epitope(s) other than known insect TRPs. Our findings indicate that at least three of the LemTRPs are always co-localized in neurons of the L. maderae brain. We have also been able to show that LemTRP-2, which is an N-terminally extended form (17-mere) of LemTRP-1 with a dibasic putative cleavage site, is transported throughout the processes of the neurons in the same manner as LemTRP-1 and 7. Thus, LemTRP-2 may be released with the other shorter LemTRPs. This is the first investigation of LemTRP distribution in the cockroach central nervous system utilizing antisera to native peptides.     

Evidence for two vitellogenin-related genes in Leucophaea maderae: the protein primary structure and its processing

We previously reported a cDNA for vitellogenin (Vg) from the cockroach, Leucophaea maderae (Lm). In the present study, we identified another cDNA encoding a second Vg (Vg2) having stretches of amino acid sequences different from the first one, Vg1, reported earlier. The complete nucleotide sequence of Vg2 consisted of 5,915 bp, which encoded a primary protein of 1,911 residues including a 16-residue putative signal peptide. The regions different in both Vg precursors (Pro-Vg1 and pro-Vg2) were four in number, and two, relatively longer, existed at the carboxy terminal. The presence of two Vg-related cDNAs was confirmed by sequencing of RT-PCR products generated using primers designed based on the common sequences flanking the regions different in amino acid sequences. Both forms were transcribed since they could be amplified on mRNA from fat bodies of different individual females. Southern blot analysis of digested genomic DNA revealed the existence of two Vg-related genes in L. maderae indicating that each Vg cDNA originated from a separate gene. Also, the immunoblot analysis using antibodies generated against peptides unique to both Vg1 and Vg2 probed the same antigen in the same individual, suggesting LmVg to be a product coded by two different Vg precursors. Both Vg primary products showed 96% similarity at an amino acid level. Compared to other insect Vgs, Vg2 showed a slightly higher (1-2%) similarity than Vg1. We previously reported, based on amino-terminal sequence analysis, that L. maderae pro-Vg was cleaved into four subunit polypeptides (112-, 100-, 92-, and 55-kD), which were deposited in the egg as four respective vitellin (Vn) polypeptides. We show now based on immunoblot analysis that the 112-kD polypeptide is further cleaved, near the C-terminus, to an 87-kD polypeptide before it is secreted into the hemolymph. Both the L. maderae Vgs were compared with each other and with other insect Vgs and the processing pattern is discussed.     

Toxicity, sublethal effects, and performance of sulfluramid against the German cockroach (Dictyoptera: Blattellidae)

Topical and oral toxicity of sulfluramid (N-ethyl perfluorooctane sulfonamide) were determined for the German cockroach, Blattella germanica (L.). The topical LD50 of sulfluramid was 175.6 micrograms/g for adult males, 117.8 micrograms/g for adult females, and 122.3 micrograms/g for gravid adult females. Ingestion increased toxicity approximately 1.4 times for adult male B. germanica. Twenty-four hours after topical treatment with 20 micrograms/insect sulfluramid, the percentage of female cockroaches that dropped their oothecae increased approximately 50% compared with controls treated with acetone. Sulfluramid also decreased oothecal hatch of both dropped and retained oothecae. Approximately 90% of oothecae from untreated females hatches, whereas less than 20% hatched from females treated with 20 micrograms/insect. Mean time for oothecal hatch increased linearly with increasing sulfluramid concentration. In arena studies in Ebeling choice boxes, LT50's ranged between approximately 2.3 and 3.9 d for a 0.331 mg/cm2 deposit and a 1.5% bait, respectively. Higher concentrations of sulfluramid were more repellent in both bait and residual formulations. Performance index values indicated excellent potential field efficacy. Field trials with 1.0 and 1.5% (AI) baits showed up to a 71.3% reduction in cockroach numbers. Baits controlled cockroaches throughout the 12-wk test.     

Isolation and partial characterization of five myotropic peptides present in head extracts of the cockroach Leucophaea maderae

Five peptides were isolated by reverse-phase HPLC from head extracts of the cockroach Leucophaea maderae. Four of the peptides were inactivated by aminopeptidase M (APM). The inability of APM to digest the fifth peptide suggests a blocked NH2-terminus. Four of the peptides were inactivated by carboxypeptidase Y (CPY). The activity of the fraction which would have contained proctolin was decreased by about 20%. The complete deactivation of proctolin by CPY indicated that a second peptide, co-eluting with proctolin but refractory to CPY digestion, was responsible for 80% of the biological activity in that fraction. Concentrations of the peptides necessary to produce a threshold response from the isolated cockroach hindgut ranged from 0.009 to 0.083 head equivalents/ml.