RNAi of yellow-y, required for normal cuticle pigmentation, impairs courtship behavior and oviposition in the German cockroach (Blattella germanica).

The insect cuticle plays a key role in maintaining the insect's physiological function and behavior. Herein, the yellow-y protein is required to produce black melanin, and is expressed in a pattern that correlates with the distribution of this pigment. However, yellow-y can also have other functions, for instance, in insect behavior, but not much is known. In this study, we have studied the yellow-y gene in one important model and pest species, namely the German cockroach (Blattella germanica), which is to our knowledge the first time reported. In essence, we identified the yellow-y gene (BgY-y) and characterized its function by using RNA interference (RNAi). Silencing of BgY-y gene led to different developmental abnormalities (body weight and wings) in both genders. Specifically, there was an abundant decrease in melanin, turning the body color in pale yellow and the cuticle softer and more transparent. Interestingly, we also observed that the knockdown of BgY-y impaired the male cockroaches to display a weaker response to female-emitted contact sex pheromones, and also that the oviposition ability was weakened in the RNAi females. This study comprehensively analyzed the biological functions of the yellow-y gene in German cockroaches from the perspectives of development, body color, courtship behavior and oviposition, and as a consequence, this may opens new avenues to explore it as a novel pest control gene.

Nutritional condition affects tergal gland secretion and courtship success of male cockroaches.

An integral part of the courtship sequence of the German cockroach (Blattella germanica) involves the male raising his wings to expose tergal glands on his dorsum. When a female cockroach feeds on the secretion of these glands, she is optimally positioned for mating. Core chemical components have been identified, but the effect of male diet on the quality of the tergal gland secretion remains unexplored. After validating the pivotal role of tergal feeding in mating, we starved or fed reproductively mature males for one week. We then paired each male with a sexually receptive female and observed their interactions through an infrared-sensitive camera. While starvation had no effect on male courtship behavior, it did influence the duration of female tergal feeding and mating outcomes. Females fed longer on the gland secretion of fed males, and fed males experienced greater mating success than starved males (73.9% vs. 48.3%, respectively). These results suggest that the quality of the tergal gland secretions, and by association mating success, are dependent on the nutritional condition of the male.

Modulation of fatty acid elongation in cockroaches sustains sexually dimorphic hydrocarbons and female attractiveness.

Insect cuticular hydrocarbons (CHCs) serve as important intersexual signaling chemicals and generally show variation between the sexes, but little is known about the generation of sexually dimorphic hydrocarbons (SDHCs) in insects. In this study, we report the molecular mechanism and biological significance that underlie the generation of SDHC in the German cockroach Blattella germanica. Sexually mature females possess more C29 CHCs, especially the contact sex pheromone precursor 3,11-DimeC29. RNA interference (RNAi) screen against the fatty acid elongase family members combined with heterologous expression of the genes in yeast revealed that both BgElo12 and BgElo24 were involved in hydrocarbon (HC) production, but BgElo24 is of wide catalytic activities and is able to provide substrates for BgElo12, and only the female-enriched BgElo12 is responsible for sustaining female-specific HC profile. Repressing BgElo12 masculinized the female CHC profile, decreased contact sex pheromone level, and consequently reduced the sexual attractiveness of female cockroaches. Moreover, the asymmetric expression of BgElo12 between the sexes is modulated by sex differentiation cascade. Specifically, male-specific BgDsx represses the transcription of BgElo12 in males, while BgTra is able to remove this effect in females. Our study reveals a novel molecular mechanism responsible for the formation of SDHCs and also provide evidences on shaping of the SDHCs by sexual selection, as females use them to generate high levels of contact sex pheromone.

Behavioral and Morphological Studies of the Membranous Tergal Structure of Male Blattella germanica (Blattodea: Ectobiidae) During Courtship.

The male German cockroach Blattella germanica (L.) exhibits a characteristic courtship display by raising its wings, turning around, and exposing its tergal glands on the seventh and eighth tergites to the female. The male secretes a courtship pheromone from the tergal glands which induces a strong feeding response in the female, facilitating copulation. Upon multiple, detailed microscopic observations of the courtship display, we found that the male markedly expanded the intersegmental area between the sixth and seventh tergites, but deflated this area as soon as it perceived a tactile stimulus on its back by the female, while continuing to raise its wings and exposing the tergal glands. The intersegmental area is composed of two parts: a membranous posterior part of the sixth tergite, and the regular intertergal membrane. The membranous posterior part was found to be crescentic in shape and clearly separated from the intertergal membrane by traverse tongue-shaped plates. Scanning electron microscopic observation revealed that there were many orifices or pores in the membranous crescentic zone, and its morphological structure was clearly different from that of the intertergal membrane. Our observations suggest that the crescentic zone is likely a secretory gland that may play an important role at the beginning of the male courtship display, different from the tergal glands.

Antennal grooming facilitates courtship performance in a group-living insect, the German cockroach Blattella germanica.

The antennae of adult male German cockroaches detect a contact sex pheromone embedded in the female's cuticular lipids. The female pheromone stimulates courtship behavior in males, notably a wing-raising (WR) display. Within aggregations, however, cuticular lipids are disseminated by contact among group members, including nymphs and adults of both sexes, and "contamination" of cockroaches with the cuticular lipids of another stage or sex may interfere with sex discrimination and disrupt courtship. We used behavioral observations, bioassays and chemical analysis to determine how males maintain their sensitivity to sex pheromone in aggregations. Males contaminated with female pheromone displayed lower courtship, because residual female pheromone on their antennae adapted their peripheral sensilla and habituated the central nervous system. Female pheromone that contaminated the male's antennae also elicited courtship from other non-contaminated males, disrupting their sex discrimination in the group. However, antennal grooming effectively removed female pheromone from males' antennae and maintained their chemosensory acuity and sexual discrimination among group members. Thus, grooming of the antennae and other sensory appendages is an important strategy to enhance sensory acuity, especially in group-living insects like the German cockroach.

Myoglianin triggers the premetamorphosis stage in hemimetabolan insects.

Insect metamorphosis is triggered by a decrease in juvenile hormone (JH) in the final juvenile instar. What induces this decrease is therefore a relevant question. Working with the cockroach Blattella germanica, we found that myoglianin (Myo), a ligand in the TGF-ß signaling pathway, is highly expressed in the corpora allata (CA, the JH-producing glands) and the prothoracic gland [(PG), which produce ecdysone] during the penultimate (fifth) nymphal instar (N5). In the CA, high Myo levels during N5 repress the expression of juvenile hormone acid methyl transferase, a JH biosynthesis gene. In the PG, decreasing JH levels trigger gland degeneration, regulated by the factors Krüppel homolog 1, FTZ-F1, E93, and inhibitor of apoptosis-1. Also in the PG, a peak of myo expression in N5 indirectly stimulates the expression of ecdysone biosynthesis genes, such as neverland, enhancing the production of the metamorphic ecdysone pulse in N6. The Myo expression peak in N5 also represses cell proliferation, which can enhance ecdysone production. The data indicate that Myo triggers the premetamorphic nymphal instar in B. germanica and possibly in other hemimetabolan insects.-Kamsoi, O., Belles, X. Myoglianin triggers the premetamorphosis stage in hemimetabolan insects.

Disruption of the microbiota affects physiological and evolutionary aspects of insecticide resistance in the German cockroach, an important urban pest.

The German cockroach, Blatella germanica, is a common pest in urban environments and is among the most resilient insects in the world. The remarkable ability of the German cockroach to develop resistance when exposed to toxic insecticides is a prime example of adaptive evolution and makes control of this insect an ongoing struggle. Like many other organisms, the German cockroach is host to a diverse community of symbiotic microbes that play important roles in its physiology. In some insect species, there is a strong correlation between the commensal microbial community and insecticide resistance. In particular, several bacteria have been implicated in the detoxification of xenobiotics, including synthetic insecticides. While multiple mechanisms that mediate insecticide resistance in cockroaches have been discovered, significant knowledge gaps still exist in this area of research. Here, we examine the effects of altering the microbiota on resistance to a common insecticide using antibiotic treatments. We describe an indoxacarb-resistant laboratory strain in which treatment with antibiotic increases susceptibility to orally administered insecticide. We further reveal that this strains harbors a gut microbial community that differs significantly from that of susceptible cockroaches in which insecticide resistance is unaffected by antibiotic. More importantly, we demonstrate that transfer of gut microbes from the resistant to the susceptible strain via fecal transplant increases its resistance. Lastly, our data show that antibiotic treatment adversely affects several reproductive life-history traits that may contribute to the dynamics of resistance at the population level. Together these results suggest that the microbiota contributes to both physiological and evolutionary aspects of insecticide resistance and that targeting this community may be an effective strategy to control the German cockroach.

Hemimetabolous genomes reveal molecular basis of termite eusociality.

Around 150 million years ago, eusocial termites evolved from within the cockroaches, 50 million years before eusocial Hymenoptera, such as bees and ants, appeared. Here, we report the 2-Gb genome of the German cockroach, Blattella germanica, and the 1.3-Gb genome of the drywood termite Cryptotermes secundus. We show evolutionary signatures of termite eusociality by comparing the genomes and transcriptomes of three termites and the cockroach against the background of 16 other eusocial and non-eusocial insects. Dramatic adaptive changes in genes underlying the production and perception of pheromones confirm the importance of chemical communication in the termites. These are accompanied by major changes in gene regulation and the molecular evolution of caste determination. Many of these results parallel molecular mechanisms of eusocial evolution in Hymenoptera. However, the specific solutions are remarkably different, thus revealing a striking case of convergence in one of the major evolutionary transitions in biological complexity.

Deltamethrin-resistant German Cockroaches Are Less Sensitive to the Insect Repellents DEET and IR3535 than Non-resistant Individuals.

The German cockroach, Blattella germanica (L.) (Blattodea: Blattellidae), is a serious worldwide pest with a considerable economical and sanitary impact. It is mainly controlled by the application of synthetic insecticides, but repeated use of these substances has promoted the appearance of resistance in cockroach populations throughout the world. The aim of this study was to compare the behavior of deltamethrin-susceptible (CIPEIN colony) and deltamethrin-resistant (JUBA and VGBA colonies) first instar nymphs exposed to the repellents N,N-diethyl-3-methylbenzamide (DEET) and ethyl 3-[acetyl(butyl)amino]propanoate (IR3535). Firstly, the behavior of the nymphs was assessed in an experimental arena in the absence of repellents. The parameters Distance Traveled, Velocity, Mobility Time, and Time Spent (in each half of the arena) were quantified using an image analyser, and showed that the behavior elicited by the three colonies was similar. After this, the behavior of the nymphs was quantified in an arena, half of which had been treated with repellent. The repellency of DEET increased as a linear function of log concentration for the three colonies. DEET elicited repellency as from a concentration of 97.49 µg/cm2 for the CIPEIN and JUBA colonies and 194.98 µg/cm2 for the VGBA colony. The repellency of IR3535 was weaker and started at a concentration of 389.96 µg/cm2 for the CIPEIN colony, 779.92 µg/cm2 for JUBA, and 1559.84 µg/cm2 for VGBA . Finally, nymphs were exposed to 3:1, 1:1, and 1:3 DEET:IR3535 mixtures, and a synergistic effect was observed only in the CIPEIN colony.

RNA interference and functional characterization of a tergal gland alpha amylase in the German cockroach, Blattella germanica L.

German cockroach males possess tergal glands that secrete a combination of oligosaccharides, lipids and proteins. Four major proteins occur in the secretion, with one being the 63 kDa alpha-amylase Blattella germanica Tergal Gland protein-1 (BGTG-1). Denaturing and starch gel electrophoresis coupled with peptide sequencing verified amylase activity for the BGTG-1 protein. BGTG-1 gene expression profiles were determined by using quantitative real-time PCR to compare messenger RNA abundance among isolated tissues of males, females and gravid females. Differences in BGTG-1 gene expression occurred among male tissues, with tergal gland tissue showing the highest expression. Tissues of nongravid and gravid females had significantly lower expression in comparison with male tergal glands (gravid females lowest). RNA interference (RNAi) was used to silence BGTG-1 gene expression by injecting BGTG-1 homologous double-stranded RNA (dsRNA) into male cockroaches. Groups injected with BGTG-1 dsRNA showed ∼90% lower BGTG-1 gene and protein expression compared to controls, which correlated with lower amylase activity in colorimetric assays. However, behavioural assays comparing precopulatory behaviour and mating success between RNAi and control males did not reveal differences. These results connect amylase gene expression and activity in tergal gland tissue but suggest other factors, such as other tergal gland components, may contribute more strongly to mating success.

New Food Baits for Trapping German Cockroaches, Blattella germanica (L.) (Dictyoptera: Blattellidae).

German cockroaches (GCRs), Blattella germanica (L.) (Dictyoptera: Blattellidae), are attracted to those beer semiochemicals (e.g., ethanol) that formerly living and active yeasts have produced or otherwise formed in the brewing process. We predicted that an earlier step in the production of beer, where yeasts actively metabolize the sugar in malted barley powder (dry malt extract [DME]), is very attractive to GCRs. In laboratory experiments, a 3-component composition (3CC) comprising DME, water, and Brewer's yeast strongly attracted GCR nymphs, females, and males. Both Brewers' yeast and 'spoilage organisms' in the DME or water seem to add to the attractiveness of the 3CC, but there is no additive or synergistic effect between them. The 3CC becomes optimally attractive to GCRs after 12 h of fermentation and stays that attractive for at least 120 h. In field trapping experiments, the 3CC and-unexpectedly-also the DME each proved as effective for attracting and capturing GCRs as a commercial cockroach bait (Combat Roach Gel). Future studies will investigate lethal biocontrol agents that can be added to the 3CC, or the DME, and will explore the efficacy of such lethal baits for GCR control.

Unidirectional Cross-Resistance in German Cockroach (Blattodea: Blattellidae) Populations Under Exposure to Insecticidal Baits.

Insect pests, including the German cockroach, Blattella germanica (L.), are prone to the development of physiological resistance when exposed to a number of insecticide sprays, and cross-resistance is frequently observed. Toxic baits are often used as a primary method of controlling German cockroaches, also resulting in heavy selection pressure from insecticidal baits. In response to this pressure, cockroach populations have developed aversion to specific inert ingredients in bait. Here, we examined the effect of exposure to baits containing fipronil, indoxacarb, or hydramethylnon on the development of physiological resistance to the same and other insecticides in a number of German cockroach strains. We found that prolonged exposure to baits containing fipronil or indoxacarb increased physiological resistance to these compounds. However, no increase in physiological resistance against any insecticide was observed in response to exposure to hydramethylnon bait. Additionally, we found that exposure to fipronil bait increased cross-resistance to indoxacarb. On the other hand, exposure to indoxacarb bait did not increase cross-resistance to fipronil. Neither fipronil nor indoxacarb bait exposure increased resistance to hydramethylnon. Interestingly, the development of insecticide resistance in response to bait exposure was strain-dependent and influenced by bait palatability. Our results demonstrate that exposure to toxic baits, particularly those containing fipronil, plays a significant role in the development of insecticide resistance, including cross-resistance, in German cockroaches. Further, although insecticide resistance in response to baits is mediated by exposure through the oral route, the molecular mechanisms at play are likely different for each insecticide.

Effects of foraging distance on macronutrient balancing and performance in the German cockroach Blattella germanica.

The German cockroach (Blattella germanica) is an excellent model omnivore for studying the effect of foraging effort on nutrient balancing behavior and physiology, and its consequences for performance. We investigated the effect of foraging distance on individual German cockroaches by providing two foods differing in protein-to-carbohydrate ratio at opposite ends of long containers or adjacent to each other in short containers. Each food was nutritionally imbalanced, but the two foods were nutritionally complementary, allowing optimal foraging by selective feeding from both foods. We measured nutrient-specific consumption in fifth instar nymphs and newly eclosed females foraging at the two distances, hypothesizing that individuals foraging over longer distance would select more carbohydrate-biased diets to compensate for the energetic cost of locomotion. We then determined dry mass growth and lipid accumulation in the nymphs as well as mass gain and the length of basal oocytes in the adult females as an estimate of sexual maturation. Nymphs foraging over longer distance accumulated less lipid relative to total dry mass growth, but contrary to our predictions, their protein intake was higher and they accumulated more structural mass. In concordance, adult females foraging over longer distance gained more body mass and matured their oocytes faster. Our results show a positive effect of foraging distance on fitness-related parameters at two life stages, in both cases involving increased consumption of specific nutrients corresponding to requirements at the respective life stage.

Insecticide resistance and nutrition interactively shape life-history parameters in German cockroaches.

Fitness-related costs of evolving insecticide resistance have been reported in a number of insect species, but the interplay between evolutionary adaptation to insecticide pressure and variable environmental conditions has received little attention. We provisioned nymphs from three German cockroach (Blattella germanica L.) populations, which differed in insecticide resistance, with either nutritionally rich or poor (diluted) diet throughout their development. One population was an insecticide-susceptible laboratory strain; the other two populations originated from a field-collected indoxacarb-resistant population, which upon collection was maintained either with or without further selection with indoxacarb. We then measured development time, survival to the adult stage, adult body size, and results of a challenge with indoxacarb. Our results show that indoxacarb resistance and poor nutritional condition increased development time and lowered adult body size, with reinforcing interactions. We also found lower survival to the adult stage in the indoxacarb-selected population, which was exacerbated by poor nutrition. In addition, nutrition imparted a highly significant effect on indoxacarb susceptibility. This study exemplifies how poor nutritional condition can aggravate the life-history costs of resistance and elevate the detrimental effects of insecticide exposure, demonstrating how environmental conditions and resistance may interactively impact individual fitness and insecticide efficacy.

Diet quality affects bait performance in German cockroaches (Dictyoptera: Blattellidae).

BACKGROUND: Bait formulations are widely used to control German cockroach (Blattella germanica) populations. To perform optimally, these formulations must compete favorably with non-toxic alternative foods present within the insect's habitat. We hypothesized that the nutritional history of cockroaches and their acceptance or avoidance of glucose would affect their food preference and thus bait efficacy. To test this hypothesis, we conducted a controlled laboratory experiment, first providing glucose-accepting and glucose-averse cockroaches nutritionally defined diets and then offering them identical diets containing the insecticide hydramethylnon as a bait proxy to evaluate the effect of diets of differing macronutrient composition on bait performance. RESULTS: The interaction between diet composition and bait composition affected the survival of adult males as well as first-instar nymphs exposed to excretions produced by these males. Survival analyses indicated different responses of glucose-averse and glucose-accepting insects, but generally any combination of diet and bait that resulted in high diet intake and low bait intake reduced secondary kill. CONCLUSIONS: This study represents a comprehensive examination of the effect of alternative foods on bait efficacy. We suggest that disparities between the nutritional quality of baits and the foods that are naturally available could profoundly impact the management of German cockroach infestations. © 2016 Society of Chemical Industry.

Evaluation of sublethal effects of polymer-based essential oils nanoformulation on the german cockroach.

The German cockroach, Blattella germanica (L.), is a serious household and public health pest worldwide. The aim of the present study was to evaluate the sublethal activity of polymer-based essential oils (EOs) nanoparticles (NPs) on adults of B. germanica. The LC50 and LC25 for contact toxicity were determined. To evaluate the repellency of EOs and NPs at LC25, a software was specially created in order to track multiple insects on just-recorded videos, and generate statistics using the obtained information. The effects of EOs and NPs at LC25 and LC50 on the nutritional physiology were also evaluated. The results showed that NPs exerted sublethal effects on the German cockroach, since these products enhance the repellent effects of the EOs and negatively affected the nutritional indices and the feeding deterrence index.

Gold-nanoparticles ingestion disrupts reproduction and development in the German cockroach.

The present work shows the effects of gold nanoparticles (AuNPs) orally administered on reproduction and development of the insect Blattella germanica. Newly emerged females were provided with food containing AuNPs (87.44µg/g) of a size between 15 and 30nm (mean 21.8nm), and were allowed to mate with males. Food ingestion, mortality, reproductive parameters (time to ootheca formation and eclosion, ootheca viability and fertility) as well as postembryonic developmental parameters of the first ootheca (nymphal survival and life span) were recorded throughout the experiment. Gold from AuNPs was accumulated by adults of B. germanica with a bioaccumulation factor of 0.1. Ingestion of AuNPs did not disturb the time for ootheca formation nor ootheca eclosion. However, ootheca viability was decreased almost by 25% in AuNPs treated females in comparison to controls. At the same time the number of hatched nymphs was decreased by 32.8% (p<0.001) in AuNP group respect to control one. The postembryonic developmental parameters were also affected by AuNPs treatment, with a 35.8% of decrease (p<0.01) in number of nymphs that moulted to second and third instars and a reduction of their life span. Ingestion of AuNPs causes sublethal effects in B. germanica that compromises life-traits involved in population dynamics. B. germanica is proposed as a model species in nanotoxicological studies for urban environments.

Effect of exposure to chlorpyrifos on the cuticular and internal lipid composition of Blattella germanica males.

The results of our research on the cuticular and internal lipids of Blattella germanica males provide new information on variation in the composition of the cuticular and internal lipids of B. germanica males after exposure to the presence of the insecticide. gas chromatography and gas chromatography-mass spectrometry analyses were used to identify and quantify the cuticular and internal lipid composition in males and males exposed to insecticide. There were significantly more acids having an even number of carbon atoms in the molecule, and these were also generally in higher concentrations. The following acids were in a higher concentration: C16:0 and C18:1, C18:2, C18:0. In both males and males exposed to insecticide, 24 fatty acids ranging from C6 to C22 were determined. However, there was a significantly higher content of fatty acids in the surface lipids of B. germanica males after exposure to insecticide. Our results indicate a higher content of n-alkanes, sterols, particularly cholesterol, fatty acids, and fatty acid methyl esters in the B. germanica surface after exposure to chlorpyrifos than in males that were not exposed.

Gut bacteria mediate aggregation in the German cockroach.

Aggregation of the German cockroach, Blattella germanica, is regulated by fecal aggregation agents (pheromones), including volatile carboxylic acids (VCAs). We demonstrate that the gut microbial community contributes to production of these semiochemicals. Chemical analysis of the fecal extract of B. germanica revealed 40 VCAs. Feces from axenic cockroaches (no microorganisms in the alimentary tract) lacked 12 major fecal VCAs, and 24 of the remaining compounds were represented at extremely low amounts. Olfactory and aggregation bioassays demonstrated that nymphs strongly preferred the extract of control feces over the fecal extract of axenic cockroaches. Additionally, nymphs preferred a synthetic blend of 6 fecal VCAs over a solvent control or a previously identified VCA blend. To test whether gut bacteria contribute to the production of fecal aggregation agents, fecal aerobic bacteria were cultured, isolated, and identified. Inoculation of axenic cockroaches with individual bacterial taxa significantly rescued the aggregation response to the fecal extract, and inoculation with a mix of six bacterial isolates was more effective than with single isolates. The results indicate that the commensal gut microbiota contributes to production of VCAs that act as fecal aggregation agents and that cockroaches discriminate among the complex odors that emanate from a diverse microbial community. Our results highlight the pivotal role of gut bacteria in mediating insect-insect communication. Moreover, because the gut microbial community reflects the local environment, local plasticity in fecal aggregation pheromones enables colony-specific odors and fidelity to persistent aggregation sites.

Toxicity of entomopathogenic fungi, Beauveria bassiana and Lecanicillium muscarium against a field-collected strain of the German cockroach Blattella germanica (L.) (Dictyoptera: Blattellidae).

The German cockroach, Blattella germanica (L.) has been recognized as a serious health problem throughout the world. Control failures due to insecticide resistance and chemical contamination of environment have led some researchers focus on the other alternative strategy controls. Microbial insecticides such as those containing entomo pathogenic fungi could be of high significance. Lecanicillium muscarium and Beauveria bassiana grow naturally in soils throughout the world and act as a parasite on various arthropod species, causing white muscardine disease. Thus, these two species could be considered as entomopathogenic fungi. The current study conducted to evaluate the toxicity of Beauveria bassiana and Lecanicillium muscarium against German cockroach, Blattella germanica. Conidial formulations of L. muscarium (PTCC 5184) and B. bassiana (PTCC5197) were prepared in aqueous suspensions with Tween 20. Bioassays were performed using two methods including submersion of cockroaches in conidial suspension and baiting. Data were analyzed by Probit program and LC50 and LC90 were estimated. The obtained results indicated that both fungi species were toxic against German cockroach however; Beauveria bassiana was significantly 4.8 fold more toxic than L. muscarium against German cockroach using submersion method.