Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2

Bla g 2 is a major indoor cockroach allergen associated with the development of asthma. Antigenic determinants on Bla g 2 were analyzed by mutagenesis based on the structure of the allergen alone and in complex with monoclonal antibodies that interfere with IgE antibody binding. The structural analysis revealed mechanisms of allergen-antibody recognition through cation-π interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A/K251A/F162Y showed an ∼100-fold reduced capacity to bind IgE, while preserving the native molecular fold, as proven by x-ray crystallography. This mutant was still able to induce mast cell release. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and the CD4⁺ T-cell phenotype in peripheral blood mononuclear cell cultures. Although T-cell activating capacity was similar for the KKF mutant and Bla g 2 based on CD25 expression, the KKF mutant was a weaker inducer of the Th2 cytokine IL-13. Furthermore, this mutant induced IL-10 from a non-T-cell source at higher levels that those induced by Bla g 2. Our findings demonstrate that a rational design of site-directed mutagenesis was effective in producing a mutant with only 3 amino acid substitutions that maintained the same fold as wild type Bla g 2. These residues, which were involved in IgE antibody binding, endowed Bla g 2 with a T-cell modulatory capacity. The antigenic analysis of Bla g 2 will be useful for the subsequent development of recombinant allergen vaccines.     

A Densovirus of German Cockroach Blattella germanica: Detection,Nucleotide Sequence,and Genome Organization

A new Blattella germanica densovirus (BgDNV, Parvoviridae: Densovirinae, Densovirus) was found. Virus DNA and cockroach tissues infected with BgDNV were examined by electron microscopy. Virus particles about 20 nm in diameter were observed both in the nucleus and in the cytoplasm of infected cells. Virus DNA proved to be a linear molecule sized about 1.2 m. The complete BgDNV genome was sequenced and analyzed. Five ORF were detected: two coded for structural capsid proteins and were on one DNA strand, and three coded for regulatory proteins and were on the other strand. Potential promoters and polyadenylation signals were identified. Structural analysis was performed for terminal inverted repeats containing extended palindromes. The genome structure of BgDNV was compared with that of other Parvoviridae.     

Enzymatic Activities of Allergen Extracts from Three Species of Dust Mites and Cockroaches Commonly Found in Korean Home

Allergen extracts from dust mites and cockroaches commonly found in Korean homes were used to evaluate their enzymatic activity as they are believed to influence allergenicity. Allergen extracts were prepared from 3 dust mite species (Dermatophagoides farinae, D. pteronyssinus, and Tyrophagus putrescentiae) and 3 cockroach species (Blattella germanica, Periplaneta americana, and P. fuliginosa) maintained in the Korea National Arthropods of Medical Importance Resource Bank. Proteins were extracted in PBS after homogenization using liquid nitrogen. The activities of various enzymes were investigated using the API Zym system. No significant difference in phosphatase, lipase, or glycosidase activity was observed among the 6 allergen extracts, but much difference was observed in protease activity. Protease activity was assessed in more detail by gelatin zymography and the EnzChek assay. Extract from T. putrescentiae showed the highest protease activity, followed by those of the cockroach extracts. Extracts from D. farinae and D. pteronyssinus showed only weak protease activity. Gelatinolytic activity was detected mainly in a 30-kDa protein in D. farinae, a 28-kDa protein in D. pteronyssinus, a > 26-kDa protein in T. putrescentiae, a > 20-kDa protein in B. germanica, and a > 23-kDa protein in P. americana and P. fuliginosa. The information on various enzymatic activities obtained in this study may be useful for future studies. In particular, the strong protease activity found in cockroach extracts could contribute to sensitization to cockroach allergens, which is known to be associated with the development of asthma.     

Response of German cockroaches to a dispersant emitted by adult females

A bioassay technique was used to compare the response of six age/sex classes of German cockroaches, Blattella germanica (L.), to a dispersant emitted by adult females when crowded. Differences occurred among the classes in the rate, intensity, and apparently thresholds of their response. Adults, especially males, responded more strongly than nymphs. A greater repellency was associated with filter papers conditioned by non-oothecae than by oothecae bearing females, suggesting a higher emission by the former.

---

Résumé Des papiers filtres contaminés ont été utilisés pour comparer les réponses de six catégories (âge/sexe) de Blattella germanica à une substance antiagrégative émise par les femelles adultes groupées. Des différences apparaissent entre les catégories quant aux taux, intensité et seuils apparents des réponses. Les adultes, particulièrement les mâles, répondent plus vigoureusement que les larves. Le pouvoir répulsif des papiers filtres contitionnés par des femelles ne portant pas d'ootheque était supérieur à celui de femelles porteuses; ceci suggère une plus forte émission par les premières.

     

Cloning and expression of trypsin-encoding cDNA from Blattella germanica and its possibility as an allergen

In this study, the trypsin gene (bgtryp-1) from the German cockroach, Blattella germanica, was cloned via the immunoscreening of patients with allergies to cockroaches. Nucleotide sequence analysis predicted an 863 bp open reading frame which encodes for 257 amino acids. The deduced amino acid sequence exhibited 42-57% homology with the serine protease from dust mites, and consisted of a conserved catalytic domain (GDSGGPLV). bgtryp-1 was determined by both Northern and Southern analysis to be a 0.9 kb, single-copy gene. SDS-PAGE and Western blotting analyses of the recombinant protein (Bgtryp-1) over-expressed in Escherichia coli revealed that the molecular mass of the expressed protein was 35 kDa, and the expressed protein was capable of reacting with the sera of cockroach allergy patients. We also discussed the possibility that trypsin excreted by the digestive system of the German cockroach not only functions as an allergen, but also may perform a vital role in the activation of PAR-2.     

Response of German cockroaches to aggregation pheromone emitted by adult females

The response of early, middle and late instar (mixed sexes), adult male, and gravid and non-gravid female German cockroaches to aggregation pheromone on filter papers conditioned by gravid and non-gravid females was studied. Cockroaches were attracted to and settled on each of the conditioned papers. Classes varied in the strength of their response to conditioned papers, relative response to gravid versus non-gravid female conditioned paper, and in clustering behavior. A significantly greater attraction of all classes except middle instars and non-gravid females to paper conditioned by non-gravid females suggests that non-gravid females emit more aggregation pheromone than gravid females. Possible explanations of differences in response with age/sex class, including an enhanced attraction of males from female sex phermone, are discussed.

---

Résumé L'étude a porté sur les réponses de six groupes/âges/sexes de B. germanica à des papiers filtres imprégnés de phéromones émises par des femelles pleines ou non. Tous les groupes ont été attirés par les papiers traités, mais les attractions des stades larvaires moyens ou âgés ont été inférieures aux autres. 4 groupes sur 6 ont été plus attirés par le papier traité avec des femelles ne contenant pas d'oeufs mûrs que par les femelles pleines, ce qui suggère une plus forte émission par les femelles sans oeufs mûrs. Les stades larvaires moyens ont été les seuls à être plus attirés par le papier traité avec les femelles pleines. Les femelles ne contenant aucun oeuf ne manifestent aucune préférence. La tendance à former des amas inégaux était moins nette pour les stades larvaires moyens et âgés que pour les autres groupes. Les premiers stades ont montré une tendance très nette à se rassembler sur le même papier. Le rassemblement de mâles adultes sur du papier traité avec des femelles ne contenant pas d'oeufs peut avoir été partiellement une réponse à la phéromone sexuelle femelle qui avait imprégné le papier pendant le conditionnement.

     

Field trial on the control effect of fipronil bait against German cockroaches

A field trial on the control effect of fipronil poison bait against German cockroaches (Blatella germanica) was carried out at different restaurant types in Sinchon, Seoul, Republic of Korea. Monitoring was performed applying food baited traps for 2 days per week. Reduction rates of German cockroaches by applying fipronil baits were 90.9% at Korean restaurants, 96.4% at Chinese restaurants, and 89.4% in beer hall kitchens after 4 weeks of the treatment. Overall average of the reduction rate was 93.9%. As the natural reduction rate at untreated restaurants was 11.5% after 4 weeks, a correction of the average reduction rate by applying the Abbot formula was 93.1%.     

Genetic studies of a behavioral mutant,glucose aversion,in the German cockroach (Dictyoptera: Blattellidae)

Glucose aversion(Glu) is a naturally occurring behavioral mutant of the German cockroach,Blattella germanica. Earlier work suggested thatGlu is a semidominant autosomal trait. Further study was undertaken to place the mutant gene in the linkage map of the species and expand the information on its genetic basis. Linkage was investigated in test crosses with morphologic markers for 7 of the 11 autosomal linkage groups. Assays for amounts of glucose ingested per individual confirmed the expected low consumption ofGlu heterozygotes(Glu/+) and that continuous selection pressure on the parental strain had resulted in near- and probably complete homozygosity forGlu. Glu genotypes were identified by means of a discriminating ingestion that separated heterozygotes from wild type(Glu+). Variations in expression indicate that genetic factors influence food consumption inB. germanica. Positive results were obtained in linkage tests ofGlu with ruby eye(ru), indicating that theGlu locus lies in linkage group VIII (chromosome 9), a group notable for mutants that apparently result in the expression of latent, highly primitive development pathways.     

The occurrence of translocations in insecticide resistant and susceptible field-collected strains of the German cockroach

Insecticide resistant and susceptible field-collected strains of the German cockroach were surveyed for the occurrence of chromosome polymorphisms. Analyses were on meiotic cells of males that hatched from oothecae with unusual numbers (>5) of incompletely developed eggs (aberrant oothecae). Three males were reciprocal translocation heterozygotes. A spontaneous translocation was noted in one cell of a fourth male. The males were from insecticide resistant strains. Partially developed eggs in oothecae from outcrosses of two of the translocation herozygotes ceased development in early stages and were characterized by gross morphologic abnormalities. Chromosome abnormalities other than translocations occurred in a low percentage of males from aberrant oothecae in both resistant and susceptible strains. The only general differences between susceptible and resistant strains were that, in pooled data, the frequencies of unhatched oothecae and of aberrant oothecae were higher in the resistant than susceptible strains.     

Modeling population dynamics of two cockroach species: effects of the circadian clock, interspecific competition and pest control

The German cockroach Blattella germanica is obviously one of the most spread household pests in the world, and is now virtually impossible to sustain outside human constructions. The double-striped cockroach B. bisignata, on the other hand, is limited to Southeast Asia and mostly living in the open space, yet is able to establish in cockroach-free households, too. In this article, we develop a stage-structured population model of these two species to explore (i) whether their circadian clocks impact their long-term population dynamics, (ii) which of these species is a superior competitor, and (iii) how stringent potential pest control strategies have to be to significantly impact established populations of the German cockroach. The results of the model are as follows. Firstly, phase shifts in the light-to-dark cycle did not affect cockroach population dynamics unless males and females were out of phase and their mate finding abilities rather limited. In addition, for the hypothesized circadian clock genotypes, the shorter is the inactivity period relative to the activity one or the less arrhythmic is the population, the more viable the population is and the quicker it grows to large numbers. Secondly, the German cockroach was the superior competitor: it was able to invade and drive out established populations of the double-striped cockroach and prevent any invasion of the latter. Finally, only a significant and simultaneous reduction in a number of most sensitive German cockroach parameters resulted in species extirpation. Only carefully designed and data-based models of German (and double-striped) cockroach population dynamics can be helpful in our quest to win the fight over this unwelcome but very sturdy species.     

Discontinuous carbon dioxide release in the German cockroach, Blattella germanica (Dictyoptera: Blattellidae), and its effect on respiratory transpiration

The discontinuous gas exchange cycle (DGC) was described in the German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae) for the first time. Also, the effect of the DGC on water loss was investigated. The CO(2) emission pattern in both insecticide resistant and susceptible B. germanica varied with temperature. At 10, 15, and 20 degrees C the pattern was discontinuous. Cycle frequency increased at 25 and 30 degrees C, and at 35 degrees C the pattern became cyclic. In most DGCs, there was no clear distinction between the closed and flutter phases in both strains thus data for these phases were combined and analyzed as the interburst phase. The probability that B. germanica would breath discontinuously varied with temperature. Most cockroaches (62.8%) displayed DGCs at 10 degrees C, therefore measurement of metabolic rate and water loss was carried out at this temperature. Using repeated measures of analysis of variance, the interburst and burst V(.)(CO(2))(ml h(-1)) were not significantly different between the two strains. The variability in CO(2) emission during the interburst and burst phases over time was not significantly different from cycle to cycle or between strains. Overall metabolic rate during the entire recording was not significantly different between both strains. There was a significant difference in the duration of the interburst and burst phases between the strains. The susceptible strain had significantly longer interburst and burst phase durations during a complete DGC than the resistant strain. The interburst and burst phase durations were 5.01+/-0.19 and 6.21+/-0.13 min, respectively, for the resistant strain, whereas the durations were 7.16+/-0.37 and 6.73+/-0.17 min, respectively, for the susceptible strain. This resulted in a DGC of significantly longer duration (13.89+/-0.44 min) in the susceptible strain compared with the resistant strain (11.23+/-0.26 min). The duration of the interburst phase was significantly different from the open phase duration in the resistant strain such that during a single DGC lasting approximately 11.23 min, 43.5% consisted of the interburst phase while the burst phase made up 56.5% of the cycle. The cuticular permeability at 10 degrees C and 0% RH was 2.26 microg cm(-2) h(-1) mmHg(-1) for the resistant strain and 3.42 microg cm(-2) h(-1) mmHg(-1) for the susceptible strain. In both strains, cuticular transpiration accounted for approximately 95% of total water loss. The significantly longer duration of the interburst phase of the susceptible strain was not important in reducing water loss.     

Effects on German cockroach nymphs of contact exposure to IGRs, singly and in combination

Late instar German cockroach male and female nymphs were exposed continuously for two weeks to surfaces treated with fenoxycarb, diflubenzuron, and pyriproxyfen, singly and in combination. Concentrations were determined that eliminated or nearly eliminated reproduction in matings with untreated mates, either through mortality, effects on reproduction, or a combination of mortality and sterility (no hatch). The major effect of fenoxycarb, pyriproxyfen, and pyriproxyfen plus fenoxycarb was on reproduction. The major effect of diflubenzuron was mortality. No hatch occurred in matings of females that were exposed to low concentrations of pyriproxyfen and fenoxycarb (2 ng/cm² and 6 ng/cm², respectively); sterility was incomplete when females were exposed to 600 ng/cm² of diflubenzuron. Mortality and sterility acted together to eliminate productive matings (matings that produced nymphs) when relatively high concentrations of diflubenzuron were combined with one or both of the other insect growth regulators (IGRs). In the triple combination, very small amounts of fenoxycarb and pyriproxyfen (total 1.1 ng/cm²) combined with 200 ng/cm² of diflubenzuron eliminated productive matings of treated females, but similar results with treated males were found only at higher concentrations of each IGR.     

Spatial distribution of the cockroach, Blattella germanica, in a swimming-bath facility

A population of Blattella germanica L. was studied in a swimming-bath facility using a sampling method. A set of traps deposited weekly, over a period of 50 weeks, allowed us to follow the variations of the temporal and spatial distributions of the population inside the building. A Factorial Correspondance Analysis, performed on the data, led us to the conclusion that the population was stable in time. The spatial distribution of the insects was contiguous. Inside an aggregate, we were able to define a circular movement from the center towards the periphery and vice versa; this movement can be superimposed up on the biological development on the individuals. After birth in the middle of the aggregate, during development, larvae explore further a field as far as the border of the aggregate; this exploratory phase, related to foraging behavior or search for new shelters when these become limiting factors, could be considered as the spreading phase of the population. Then, with the imminence of the imaginal moult, old larvae tend to gather in high density areas in the middle of the aggregate. Newly molted adults look for a sexual partner. And finally females remain in the shelters during oothecae maturation.

---

Résumé L'étude d'une population de Blattella germanica L. dans une piscine par des piégeages hebdomadaires pendant une période de 50 semaines, nous a permis de suivre l'évolution spatiale et temporelle de cette population. Les résultats des piégeages étudiés par Analyse Factorielle des Correspondances nous permettent de conclure à une certaine stabilité temporelle de la population. Elle se répartit en larges agrégats dans lesquels s'inscrit un mouvement circulaire, allant du centre vers la périphérie et retour, qui se superpose au cycle de développement des individus.

     

Behavioral and electrophysiological studies on the sexually biased synergism between oligosaccharides and phospholipids in gustatory perception of nuptial secretion by the German cockroach

Females of Blattella germanica feed compulsively on a nuptial secretion from the male's eighth tergal gland (TG-8) during courtship. Using TG-8 extract and its essential ingredients, maltotriose (MT) and 1,2-dioleoylphosphatidylcholine (PC), we investigated the perception of the secretion by gustatory sensilla in both sexes. Female-biased chemosensitivity was found in the feeding responses to the TG-8 extract. The TG-8 extract induced specific impulses in four functionally different receptor cells: the sugar, salt and other two types of receptor cells in a single gustatory sensillum on the paraglossae in both sexes. The impulse frequencies of the sugar receptor cell and a receptor cell of unknown type were significantly higher in females than those in males. There were no sexual differences in the behavioral and electrophysiological responses to MT alone; no responses were elicited by PC. However, a mixture of MT and PC elicited the behavioral responses more strongly than MT in females. The impulse frequency of sugar receptor cells toward the mixture of MT and PC also increased in females but not in males. These results suggest that the synergistic effect of PC on ‘sweetness’ of MT in the female cockroach contributes to her compulsive nuptial feeding. The female-biased gustatory sensitivity seems to ensure successful coupling during the nuptial feeding.     

Phospholipid composition and pheromonal activity of nuptial secretion of the male German cockroach, Blattella germanica

As a part of the sequential courtship behavior of the German cockroach, Blattella germanica, females feed on the nuptial secretion from the male tergal glands. The pheromonal secretion, consisting mainly of oligosaccharides and phospholipids, strongly elicits a feeding response in virgin females. The phospholipids were composed of phosphatidylethanolamine and phosphatidylcholine. Their fatty acid compositions were determined by chromatographic and enzymatic methods. Although an authentic blend of phospholipids (1,2-dioleoylphosphatidylethanolamine and 1,2-dioleoylphosphatidylcholine) showed a marginal phagostimulant activity, addition of the blend to an authentic blend of oligosaccharides (maltose and maltotriose) strongly enhanced the activity to nearly the level of the crude extract. These results indicate that the nuptial feeding behavior is elicited by a synergistic action between phospholipids and oligosaccharides.     

Detection of Gnathostoma spinigerum Antibodies in Sera of Non-Traumatic Subarachnoid Hemorrhage Patients in Thailand

Gnathostoma spinigerum can cause subarachnoid hemorrhage (SAH). The detection of specific antibodies in serum against G. spinigerum antigen is helpful for diagnosis of neurognathostomiasis. There is limited data on the frequency of G. spinigerum infection in non-traumatic SAH. A series of patients diagnosed as non-traumatic SAH at the Srinagarind Hospital, Khon Kaen University, Thailand between January 2011 and January 2013 were studied. CT or MR imaging of the brain was used for diagnosis of SAH. Patients were categorized as aneurysmal subarachnoid hemorrhage (A-SAH) or non-aneurysmal subarachnoid hemorrhage (NA-SAH) according to the results of cerebral angiograms. The presence of specific antibodies in serum against 21- or 24-kDa G. spinigerum antigen was determined using the immunoblot technique. The detection rate of antibodies was compared between the 2 groups. Of the 118 non-traumatic SAH patients for whom cerebral angiogram and immunoblot data were available, 80 (67.8%) patients had A-SAH, whereas 38 (32.2%) had NA-SAH. Overall, 23.7% were positive for specific antibodies against 21- and/or 24-kDa G. spinigerum antigen. No significant differences were found in the positive rate of specific antibodies against G. spinigerum in both groups (P-value=0.350).     

Structure of the house dust mite allergen Der f 2: implications for function and molecular basis of IgE cross-reactivity

The X-ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 A resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti-parallel beta-sheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross-reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.