Role of allatostatin‐like factors from the brain of Tenebrio molitor females

The effect of brain extract from females of freshly emerged Tenebrio molitor on ovary, oocyte development, total protein content of hemolymph, and ovary was studied in 4‐day‐old adult mealworm females. Injections of extracts of 2‐brain equivalents into intact (unligatured) Tenebrio females did not affect ovarian and oocyte development. Injections of ligated females, however, with 2‐brain equivalents on day 1 and 2 after adult emergence strongly inhibited ovarian growth and oocyte development. At day 4, ligated and injected females did not develop their ovaries and pre‐vitellogenic oocytes were not found. The changes in ovarian development correlated with an increase in the concentration of soluble proteins in the hemolymph as compared with the saline‐injected controls. Additionally, a strong reduction of total protein content in ovarian tissue was observed. Reverse phase HPLC separation of a methanolic brain extract of T. molitor females showed that fraction 5 has a similar retention time to synthetic cockroach allatostatin. Fraction 5 was eluted at 12.88 min, which was closest to the internal standard Dippu‐AST I, which eluted at 12.77 min. An ELISA of fraction 5 from the methanolic brain extract using antibodies against allatostatins Grybi‐AST A1 and Grybi‐AST B1 from cricket Gryllus bimaculatus showed that fraction 5 cross‐reacted with Grybi‐AST A1 antibodies. The cross‐reactivity was similar to the synthetic allatostatin from D. punctata, which was used as a positive control. These observations demonstrate a possible role for allatostatin‐like brain factor(s) in regulating the reproductive cycle of Tenebrio molitor. © 2009 Wiley Periodicals, Inc.     

Newly identified allatostatin Bs and their receptor in the two-spotted cricket,Gryllus bimaculatus

A cDNA encoding allatostatin Bs (ASTBs) containing the W(X)₆W motif was identified using a database generated by a next generation sequencer (NGS) in the two-spotted cricket, Gryllus bimaculatus. The contig sequence revealed the presence of five novel putative ASTBs (GbASTBs) in addition to GbASTBs previously identified in G. bimaculatus. MALDI-TOF MS analyses revealed the presence of these novel and previously identified GbASTBs with three missing GbASTBs. We also identified a cDNA encoding G. bimaculatus GbASTB receptor (GbASTBR) in the NGS data. Phylogenetic analysis demonstrated that this receptor was highly conserved with other insect ASTBRs, including the sex peptide receptor of Drosophila melanogaster. Calcium imaging analyses indicated that the GbASTBR heterologously expressed in HEK293 cells exhibited responses to all identified GbASTBs at a concentration range of 10⁻¹⁰–10⁻⁵ M.     

Molecular cloning and characterization of a glycosyl hydrolase family 9 cellulase distributed throughout the digestive tract of the cricket Teleogryllus emma

A novel endogenous β-1,4-endoglucanase (EG) gene belonging to the glycosyl hydrolase family 9 (GHF 9) that is distributed throughout the digestive tract of the cricket Teleogryllus emma was cloned and characterized. This gene, named TeEG-I, consists of eight exons encoding 453 amino acid residues and exists as a single copy in the T. emma genome. TeEG-I possesses all the features, including signature motifs and catalytic domains, of GHF 9 members, sharing high levels of identity with the termite, Mastotermes darwiniensis (64% protein sequence identity), and the cockroach, Panesthia cribrata (62%), GHF 9 cellulases. Recombinant TeEG-I, which is expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells, showed an optimal pH and temperature of pH 5.0 and 40 °C. The K_m and V_max values for digestion of carboxymethyl cellulose were 5.4 mg/ml and 3118.4 U/mg, respectively. Northern and Western blot analyses revealed that TeEG-I is present throughout the digestive tract, which correlated with the TeEG-I distribution and cellulase activity in the digestive tract as assayed by immunofluorescence staining and enzyme activity assay, respectively. These results indicate that TeEG-I is distributed throughout the entire digestive tract of T. emma, suggesting a functional role of endogenous TeEG-I in a sequential cellulose digestion process throughout the T. emma digestion tract.     

Targeted gene delivery in the cricket brain,using in vivo electroporation

The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb′-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors.     

Environmental adaptation and genetic variations in geographically isolated Emma field crickets Teleogryllus emma (Orthoptera: Gryllidae)

The Emma field cricket, Teleogryllus emma (Ohmachi & Matsuura), distributed between 43°N and 30°N in the Japanese archipelago, is univoltine and overwinters in the egg stage. Its eggs hatch on the slope of the Oishi Dam (38.03°N, 139.57°E, 160–170 m a.s.l.) in late June, adults begin emerging from late August, and oviposition lasts until early October. Oviposition is limited to the period when the water level of the Oishi Dam is low. The period from egg hatching to adult emergence is approximately 1 month shorter than that of the T. emma population on the Arakawa riverside (38.09°N, 139.57°E, 29 m a.s.l.), which is approximately only 7 km from the Oishi Dam. The egg and body sizes of T. emma on the slope of the Oishi Dam were smaller than those of T. emma on the Arakawa riverside, and the egg and nymphal periods were shorter; these variations were inherited by the next generation of T. emma. The egg period, nymphal period and head width of T. emma on the dam slope correspond to those of the populations near 40°N. Several traits of the T. emma population on the dam slope were naturally selected by adapting to the isolated environment, resulting in the genetic variations. However, their variations were small and the period after isolation is short, suggesting that it is in the early stages of speciation.     

Microsatellite loci for the field cricket,Gryllus bimaculatus and their cross‐utility in other species of Orthoptera

We have isolated 16 microsatellite loci in the field cricket, Gryllus bimaculatus. Nine loci were found to be polymorphic in G. bimaculatus and the number of alleles varied from seven to 14. All 16 loci were tested for amplification in nine other species. In the five species tested belonging to the same subfamily (Gryllinae), a minimum of nine loci amplified. These loci will be used to determine paternity as part of a study to investigate the genetic benefits of polyandry.     

Adipokinetic and hyperglycaemic factors of different insect species: Separation with high performance liquid chromatography

Corpus cardiacum extracts from the phasmids, Carausius morosus, Cuniculina impigra, Sipyloidea sipylus, Acrophylla wuelfingi, Eurycantha goliath, Bacillus rossius and Extatosoma tiaratum, from the Orthopterans, Locusta migratoria and Gryllus bimaculatus, from the Dictyopterans, Periplaneta americana, Gromphadorrhina coquereliana and Blaberus craniifer, from the Coleopterans Tenebrio molitor and Pachnoda sp., synthetic adipokinetic hormone and synthetic crustacean red pigment-concentrating hormone (RPCH) were injected into locusts, cockroaches and ligated stick insects as bioassay systems for adipokinetic and hyperglycaemic substances, respectively. The locust and cockroach bioassay gave positive results with all corpus cardiacum material tested (however the lipid response in locusts upon injection of T. molitor corpus cardiacum extract was very poor). The stick insect bioassay was quite specific for stick insect corpus cardiacum material; only corpus cardiacum extracts from a few other species (G. bimaculatus, P. americana, G. coquereliana and Pachnoda sp.) showed weak activity. All other extracts, including synthetic adipokinetic hormone and RPCH, failed to induce a response.Separations of corpus cardiacum extracts from L. migratoria, P. americana, T. molitor, C. morosus and S. sipylus were achieved on reversed-phase high-performance liquid chromatography (RP-HPLC). Locust corpus cardiacum extract showed two absorbance peaks with adipokinetic activity, adipokinetic hormones I and II. The peaks with hyperglycaemic activity from P. americana corpus cardiacum extracts had different retention times to those of locust adipokinetic hormones I and II. Stick insect corpus cardiacum extracts revealed also 2 absorbance peaks with adipokinetic activity, the major one co-eluting with RPCH. The active compound from corpus cardiacum extracts of T. molitor appeared to elute close to locust adipokinetic hormone I.     

Biology and host range testing of Triarthria setipennis and Ocytata pallipes (Diptera: Tachinidae) for the control of the European earwig (Forficula auricularia)

The European earwig (Forficula auricularia) is an invasive species in the Falkland Islands, causing considerable problems for local horticulture, as well threatening indigenous ecosystems. To assess the potential of a classical biological control introduction two parasitoid fly species, Triarthria setipennis and Ocytata pallipes (Diptera: Tachinidae), were collected from sites in southern and northern England and then tested for their suitability as earwig biological controls at Egham, UK. Both species had previously been introduced into North America for earwig control however little is known of their long-term efficacy and host specificity. Host range tests including both target and non-target species were done. As there are no native Dermaptera on the Falkland Islands, tests were restricted to the field cricket Gryllus assimilis and the Dubia cockroach Blaptica dubia, as representatives of insect orders phylogenetically closely related to earwigs. A second cricket species (Gryllus bimaculatus) was included in an egg-depositing experiment for O. pallipes. Both tachinid species successfully parasitised and emerged from earwigs under laboratory conditions but no signs of parasitisation and development were observed in either the cricket or cockroach.     

Immunological evidence for FGLamide- and W2W9-allatostatins in the ovary of Gryllus bimaculatus (Ensifera, Gryllidae)

Using polyclonal antisera to Gryllus bimaculatus allatostatin A1 (FGLamide) and B1 (W²W⁹‐amide), two sensitive and specific competitive enzyme‐linked immunosorbent assays (ELISAs) were developed, which allowed detection of A‐ and B‐allatostatin‐like immunoreactivity in extracts of ovaries and partially purified chromatography (HPLC) fractions. Immuno‐positive HPLC fractions inhibited biosynthesis of Juvenile Hormone in vitro in the rapid partition assay. Both antisera were also used to immunolocalize A‐ and B‐allatostatin epitopes in the ovary of larval and adult crickets. A‐ and B‐allatostatin immunoreactivity appeared in the cortical cytoplasm of the oocytes at the anterior cell poles. Controls treated with the pre‐immune sera or with antisera preadsorbed onto the respective peptides coupled to a solid phase support did not stain. There was no staining in any neural structures within the ovary.     

An extract of Ulmus macrocarpa improves cellular immunity in immuno-suppressed models

An extract of Ulmus macrocarpa Hance, commonly known as the large-fruited elm, has been prescribed as a traditional medicine. In this study, we aimed to investigate the cellular immune effects of U. macrocarpa stem cortex extracts on cyclophosphamide (CY)-treated splenocytes and mice. A methanol extract showed an improved survival rate of splenocytes after 72?h. The extract was successively partitioned with dichloromethane, ethyl acetate, n-butanol, and water; and the fractions so obtained were also examined for their proliferative activity. Among them, the water fraction of U. macrocarpa showed the highest proliferation of splenocytes and was used throughout the present study. We tested the survival rate of splenocytes through dose-dependent treatment of CY and the suppression of the survival effect by CY was recovered by treatment with the water extract of U. macrocarpa. To determine the mechanism involved, we examined the expression of B-cell lymphoma-extra large (Bcl-xL) anti-apoptotic protein. CY decreased Bcl-xL protein levels in resting splenocyte cultures, whereas splenocytes were exposed to water extracts of U. macrocarpa in the presence of CY; however, elevations in Bcl-xL were observed at 96?h. Mice splenocytes treated with water extract of U. macrocarpa for cellular immunity showed an increase in the activity of the mixed lymphocyte reaction (MLR), cytotoxic T lymphocytes (CTLs), and natural killer (NK) cells. In addition, mice receiving a water extract of U. macrocarpa recovered the CTL, NK, and MLR activities suppressed by CY administration. Consequently, U. macrocarpa improves the cell-mediated immune response and provides an insight on cell-based tonic materials.     

Immunity of the greater wax moth Galleria mellonella

Investigation of insect immune mechanisms provides important information concerning innate immunity, which in many aspects is conserved in animals. This is one of the reasons why insects serve as model organisms to study virulence mechanisms of human pathogens. From the evolutionary point of view, we also learn a lot about host–pathogen interaction and adaptation of organisms to conditions of life. Additionally, insect‐derived antibacterial and antifungal peptides and proteins are considered for their potential to be applied as alternatives to antibiotics. While Drosophila melanogaster is used to study the genetic aspect of insect immunity, Galleria mellonella serves as a good model for biochemical research. Given the size of the insect, it is possible to obtain easily hemolymph and other tissues as a source of many immune‐relevant polypeptides. This review article summarizes our knowledge concerning G. mellonella immunity. The best‐characterized immune‐related proteins and peptides are recalled and their short characteristic is given. Some other proteins identified at the mRNA level are also mentioned. The infectious routes used by Galleria natural pathogens such as Bacillus thuringiensis and Beauveria bassiana are also described in the context of host–pathogen interaction. Finally, the plasticity of G. mellonella immune response influenced by abiotic and biotic factors is described.     

Pigment‐dispersing factor affects circadian molecular oscillations in the cricket Gryllus bimaculatus

Pigment‐dispersing factor (PDF) is an important neurotransmitter in insect circadian systems. In the cricket Gryllus bimaculatus, it affects nocturnal activity, the free‐running period and photic entrainment. In this study, to investigate whether these effects of PDF occur through a circadian molecular machinery, we measured mRNA levels of clock genes period (per) and timeless (tim) in crickets with pdf expression knocked‐down by pdf RNAi. The pdf RNAi decreased per and tim mRNA levels during the night to reduce the amplitude of their oscillation. The phase of the rhythm advanced by about 4 h in terms of trough and/or peak phases. On the other hand, pdf mRNA levels were little affected by per and tim RNAi treatment. These results suggest that PDF affects the circadian rhythm at least in part through the circadian molecular oscillation while the circadian clock has little effect on the pdf expression.     

Separation of oviposition-stimulating peptides and myotropic factors from head extracts of Galleria mellonella L.: Comparative effects of myotropic and non-myotropic factors on egg laying

Methanolic extracts from heads of the wax moth, Galleria mellonella L. contain several factors that stimulate oviposition of virgin females in vivo and spontaneous contractions of the oviduct in vitro of the cricket, Gryllus bimaculatus. The myotropic and egglaying activities behaved distinctly during all steps of purification, including fractionation on reverse-phase high-performance liquid chromatography. A novel neuropeptide was isolated from Galleria mellonella. The peptide does not increase the motility of isolated cricket oviducts. Among 13 other myotropic or non-myotropic factors studied, none were found to be as potent as the new peptide in stimulating egg-laying activity. The dose-response curves of myotropic and non-myotropic factors indicate that (i) proctolin, l-glutamate, octopamine, leucokinin-VI, leucopyrokinin, ecdysone and 20-hydroxy-ecdysone, or equivalent structures, might be involved in Galleria mellonella oviposition control, and (ii) that only the novel wax moth neuropeptide has the ability to trigger egg laying in that insect. The significance of these findings is discussed.Abbreviations AcN acetonitrile - E head equivalent - 20-HE 20-hydroxyccdysone - JH 20-hydroxyecdysone, juvenile hormone - Lem-K VI leucokinin-VI - Lem-K I leucokinin-I - Lem MS leucomyosupressin - Lem PK leukopyrokinin - TFA trifluoroacetic acid     

RESPONSE OF PARASITOID MICROPLITIS MEDIATOR TO PLANT VOLATILES IN AN OLFACTOMETER*

Abstract The behavioral responses of Microplitis mediator were measured in a four‐armed olfactometer. Leaves extract of the suitable host plant of its insect host, cotton, elicited higher olfactory responses; while leaves extract of the less suitable host plant of its insect host, tomato or tobacco, elicited moderate responses. Volatiles from the least suitable host plant of its insect host, hot pepper, elicited the lowest responses. The different preferences to four plant leaves extracts suggested that M. mediator could discriminate between suitable and less suitable host plants of its insect host by semiochemicals. Green leaf volatiles (GLVs), cis‐3‐hexen‐1‐yl acetate, hexanal, and nonan‐1‐ol, are the most attractive compound among the 7 tested compounds. The parasitoids preferred host‐damaged plant to undamaged plant, suggesting that they were able to distinguish between host and non‐host of their insect host through varying chemical blends. Experience with cotton leaves extract prior to experiment enhanced their responses to the same odor.     

Neurohormonal control of the mating interval in the male cricket,Gryllus bimaculatus DeGeer

Summary Between two mating acts of the male cricket (Gryllus bimaculatus), spermatophore protrusion (SP) and courtship stridulation (CS), there is a fixed time interval. This interval lasts about 1 h. During the period from SP to CS, the male cricket does not stridulate nor make any type of mating sound (post-spermatophore protrusion silence: PSPS) and tolerates external sensory stimuli. We examined the effects of injections of hemolymph and ganglia extracts on the interval. Extracts obtained from crickets which had just started CS (CS crickets) and those which had finished SP (SP crickets) were effective. The extracts were fractionated by ul trafiltration. Fractions with a molecular weight of less than 1 kdalton affected the length of the PSPS. The fractions from both the hemolymph and the mesothoracic ganglion of CS crickets shortened the PSPS. On the other hand, the fractions from the hemolymph and the brain of SP crickets lengthened the PSPS. We estimated, by gel filtration, the molecular weight of the effective fractions from the mesothoracic ganglion and the brain to be 100–200 daltons. We also examined the effects of biogenic amines on the PSPS. Octopamine shortened the PSPS, whereas serotonin lengthened it. The results in dicate that at least two neurohormones from the brain and the mesothoracic ganglion reciprocally control the elicitation of CS and provide an appropriate interval in the mating sequence of the male cricket. Octopamine and serotonin are possible candidates for these neurohormones.Abbreviations CP copulation - CS courtship stridulation - SP spermatophore protrusion - PSPS post-spermatophore protrusion silence     

Toxicity of black nightshade (Solanum nigrum) extracts on Alternaria brassicicola, causal agent of black leaf spot of Chinese cabbage (Brassica pekinensis)

Root extracts of black nightshade (Solanum nigrum) were analyzed for activity against isolates ABA‐31 and ABA‐104 of Alternaria brassicicola, the causal agent of black leaf spot of Chinese cabbage (Brassica pekinensis). Preliminary results showed that dried root tissues of black nightshade extracted with 70% ethanol contained antifungal properties against A. brassicicola. Ethanol root extracts were used for further fractionations using ethyl acetate, n‐butanol and water. Among the three extracts, the n‐butanol fraction showed the strongest antifungal activity by its suppression of conidial germination of A. brassicicola. The n‐butanol extract of S. nigrum roots was fractionated further into six fractions (I–VI). Among the six fractions tested, fraction V showed a strong inhibitory effect on conidial germination of A. brassicicola and thereby suppressed lesion development of black leaf spot of Chinese cabbage at a concentration of 25 ppm or higher. Nuclear magnetic resonance analysis indicated that fraction V contained a mixture of saponins, and results of further bio‐guided fractionation and bioassay suggested that saponins in fraction V were key chemical components in the control of A. brassicicola. The potential of using black nightshade for developing natural products for the control of fungal plant diseases is discussed.     

Production of chitin and chitosan from the exoskeleton of adult two‐spotted field crickets (Gryllus bimaculatus)

Chitin and chitosan were extracted from all specimens of Type I and II two‐spotted field crickets (Gryllus bimaculatus) following chemical treatment with an acid and alkali. For chitin extraction, 2 N HCl and 1.25 N NaOH solutions were used to achieve demineralization and deproteinization, respectively. For chitosan extraction, 50 % NaOH (w/v) and 50 % NaOH (w/w) solutions were used to achieve deacetylation. Chitosan yielded from adult exoskeletons of G. bimaculatus in Test A of Type I was 1.76 and 8.40 % on a fresh weight (FW) and dry weight (DW) basis, respectively, after treatment with 50 % NaOH (w/v) at 95°C for 3 h. Furthermore, the chitosan yielded in Test D of Type II was 1.79 and 7.06 % on FW and DW basis, respectively, after treatment with 50 % NaOH (w/w) at 105°C for 3 h. The average yield of chitin and chitosan was 2.42 and 1.65 % on a FW basis, and 10.91 and 7.50 % on DW basis, respectively. The deacetylation (%) of chitosan extracted from adult exoskeletons in Tests A, B, C1, C2, D1, and D2 were 81.2 %, 14.5 %, 19.6 %, 90.7 %, 17.1 %, and 95.5 %, respectively. The viscosities of the chitosans extracted from adult exoskeletons in Tests A, C2, and D2 were 32.0, 21.6, and 62.4 cP (centi Poise), respectively. The molecular weight of chitosan from adult exoskeletons of G. bimaculatus was 308.3 kDa. Our results indicate that adult exoskeletons of G. bimaculatus could be used as a source of chitin and chitosan for use as functional additives in industrial animal feeds.     

Fine‐ and Broad‐Scale Approaches to Understanding the Evolution of Aggression in Crickets

Male field crickets frequently engage in agonistic contests to establish dominance in social interactions and gain access to mate attraction territories. Crickets (Orthoptera: Gryllidae) are often used as a model taxon to study aggression, but limited documentation of aggression in some cricket species hinders our understanding of its evolutionary costs and benefits. Our study investigated cricket aggression at two scales: the within‐species scale for two cricket species, Gryllus assimilis and G. veletis, whose aggression had not been adequately documented and the among‐species scale to detect evolutionary patterns in species’ levels of aggression. In both G. veletis and G. assimilis, winners spent more time being aggressive than losers, but they were not larger or heavier. Collectively, our results reveal that G. veletis males are more aggressive than G. assimilis. Male G. veletis had higher aggression scores that male G. assimilis. The majority of G. veletis contests escalated to grappling (a highly aggressive behavior), while less than one quarter of G. assimilis contests escalated to grappling. Further, G. veletis males transitioned between two of the most aggressive behaviors most often while G. assimilis transitioned between two of the least aggressive behaviors most often. We integrate this new information on aggression for G. assimilis and G. veletis with previously documented aggression data for many cricket species to investigate aggression in a broader evolutionary context than previously possible. Within a phylogenetic context, we test the hypothesis that species whose males use burrows from which to call and attract females are more aggressive than species with non‐burrowing males. We found evidence consistent with this hypothesis; species with burrowing males tended to be more aggressive than species with non‐burrowing males. Together, our study provides fine‐scale understanding of aggression in two cricket species and broad‐scale evolutionary context for aggression across cricket species.