Hygiene-Eliciting Brood Semiochemicals as a Tool for Assaying Honey Bee (Hymenoptera: Apidae) Colony Resistance to Varroa (Mesostigmata: Varroidae).

Despite numerous interventions, the ectoparasitic mite Varroa (Varroa destructor Anderson and Trueman [Mesostigmata: Varroidae]) and the pathogens it vectors remain a primary threat to honey bee (Apis mellifera Linnaeus [Hymenoptera: Apidae]) health. Hygienic behavior, the ability to detect, uncap, and remove unhealthy brood from the colony, has been bred for selectively for over two decades and continues to be a promising avenue for improved Varroa management. Although hygienic behavior is expressed more in Varroa-resistant colonies, hygiene does not always confer resistance to Varroa. Additionally, existing Varroa resistance selection methods trade efficacy for efficiency, because those achieving the highest levels of Varroa resistance can be time-consuming, and thus expensive and impractical for apicultural use. Here, we tested the hypothesis that hygienic response to a mixture of semiochemicals associated with Varroa-infested honey bee brood can serve as an improved tool for predicting colony-level Varroa resistance. In support of our hypothesis, we demonstrated that a mixture of the compounds (Z)-10-tritriacontene, (Z)-8-hentriacontene, (Z)-8-heptadecene, and (Z)-6-pentadecene triggers hygienic behavior in a two-hour assay, and that high-performing colonies (hygienic response to ≥60% of treated cells) have significantly lower Varroa infestations, remove significantly more introduced Varroa, and are significantly more likely to survive the winter compared to low-performing colonies (hygienic response to <60% of treated cells). We discuss the relative efficacy and efficiency of this assay for facilitating apiary management decisions and selection of Varroa-resistant honey bees, as well as the relevance of these findings to honey bee health, pollination services, and social insect communication.

Adaptive contraction of diet breadth affects sexual maturation and specific nutrient consumption in an extreme generalist omnivore.

Animals balance their intake of specific nutrients, but little is known about how they do so when foraging in an environment with toxic resources and whether toxic foods promote adaptations that affect life history traits. In German cockroach (Blattella germanica) populations, glucose aversion has evolved in response to glucose-containing insecticidal baits. We restricted newly eclosed glucose-averse (GA) and wild-type (WT) female cockroaches to nutritionally defined diets varying in protein-to-carbohydrate (P : C) ratio (3 : 1, 1 : 1, or 1 : 3) or gave them free choice of the 3 : 1 and 1 : 3 diets, with either glucose or fructose as the sole carbohydrate source. We measured consumption of each diet over 6 days and then dissected the females to measure the length of basal oocytes in their ovaries. Our results showed significantly lower consumption by GA compared to WT cockroaches when restricted to glucose-containing diets, but also lower fructose intake by GA compared to WT cockroaches when restricted to high fructose diets or given choice of fructose-containing diets. Protein intake was regulated tightly regardless of carbohydrate intake, except by GA cockroaches restricted to glucose-containing diets. Oocyte growth was completely suppressed in GA females restricted to glucose-containing diets, but also significantly slower in GA than in WT females restricted to fructose-containing diets. Our findings suggest that GA cockroaches have adapted to reduced diet breadth through endocrine adjustments which reduce requirements for energetic fuels. Our study illustrates how an evolutionary change in the chemosensory system may affect the evolution of other traits that govern animal life histories.

Extensive Mitochondrial Heteroplasmy in Natural Populations of a Resurging Human Pest, the Bed Bug (Hemiptera: Cimicidae).

Homoplasmy, the occurrence of a single mitochondrial DNA haplotype within an individual, has been the accepted condition across most organisms in the animal kingdom. In recent years, a number of exceptions to this rule have been reported, largely due to the ease with which single nucleotide polymorphisms can be detected. Evidence of heteroplasmy-two or more mitochondrial variants within a single individual-has now been documented in a number of invertebrates; however, when present, heteroplasmy usually occurs at low frequencies both within individuals and within populations. The implications of heteroplasmy may be far reaching, both to the individual in relation to its health and fitness, and when considering the evolutionary dynamics of populations. We present novel evidence for frequent mtDNA heteroplasmy in the bed bug, Cimex lectularius L. (Hemiptera: Cimicidae). Our findings show that heteroplasmy is common, with 5 of 29 (17%) populations screened exhibiting two mitochondrial variants in a ∼1:2 ratio within each individual. We hypothesize that the mechanism underlying heteroplasmy in bed bugs is paternal leakage because some haplotypes were shared among unrelated populations and no evidence for nuclear mitochondrial DNA sequences was detected.

Diet specialization in an extreme omnivore: nutritional regulation in glucose-averse German cockroaches.

Organisms have diverse adaptations for balancing dietary nutrients, but often face trade-offs between ingesting nutrients and toxins in food. While extremely omnivorous cockroaches would seem excluded from such dietary trade-offs, German cockroaches (Blattella germanica) in multiple populations have rapidly evolved a unique dietary specialization - an aversion to glucose, the phagostimulant in toxic baits used for pest control. We used factorial feeding experiments within the geometric framework to test whether glucose-averse (GA) cockroaches with limited access to this critical metabolic fuel have compensatory behavioural and physiological strategies for meeting nutritional requirements. GA cockroaches had severely constrained intake, fat and N mass, and performance on glucose-based diets relative to wild-type (WT) cockroaches and did not appear to exhibit digestive strategies for retaining undereaten nutrients. However, a GA × WT 'hybrid' had lower glucose aversion than GA and greater access to macronutrients within glucose-based diets - while still having lower intake and survival than WT. Given these intermediate foraging constraints, hybrids may be a reservoir for this maladaptive trait in the absence of positive selection and may account for the rapid evolution of this trait following bait application.

[The intracellular localization of the regulatory proteins of the densovirus of German cockroach, Blattella germanica].

The intracellular localization of the regulatory proteins encoded by the genome of the densovirus of German cockroach was analyzed using western-blotting of nuclear and cytoplasmic extracts of the densovirus-infected passaging cells tissue culture BGE-2. Two of the three regulatory proteins, NS1 and NS3, were shown to possess mainly nuclear localization, while NS2 protein was distributed between the nucleus and cytoplasm. Data obtained provide new information necessary for prediction of the functions of densovirus regulatory proteins. Intracellular localization of NS3 protein was described for the densoviruses for the first time.

Deployment and transcriptional evaluation of nitisinone, an FDA-approved drug, to control bed bugs.

Bed bugs are blood-feeders that rapidly proliferate into large indoor infestations. Their bites can cause allergies, secondary infections and psychological stress, among other problems. Although several tactics for their management have been used, bed bugs continue to spread worldwide wherever humans reside. This is mainly due to human-mediated transport and their high resistance to several classes of insecticides. New treatment options with novel modes of action are required for their control. In this study, we evaluated the use of nitisinone (NTBC), an FDA-approved drug, for bed bug control in an insecticide-susceptible (HH) and an insecticide-resistant (CIN) population. Although NTBC was lethal to both populations when administered orally or applied topically in very low doses, we observed a slight but significant resistance in the CIN population. Transcriptomic analysis in both populations indicated that NTBC treatment elicited a broad suppression of genes associated with RNA post-transcriptional modifications, translation, endomembrane system, protein post-translational modifications and protein folding. The CIN population exhibited higher ATP production and xenobiotic detoxification. Feeding studies on a mouse model highlight that NTBC could be used as a control method of bed bugs by host treatment. The results demonstrate that NTBC can be used as a new active ingredient for bed bug control by topical or oral treatment and shed light on the molecular mechanisms of suppressed tyrosine metabolism following NTBC treatment.

One quantitative trait locus for intra- and interspecific variation in a sex pheromone.

Even though premating isolation is hypothesized to be a major driving force in speciation, its genetic basis is poorly known. In the noctuid moth Heliothis subflexa, one group of sex pheromone components, the acetates, emitted by the female, plays a crucial isolating role in preventing interspecific matings to males of the closely related Heliothis virescens, in which females do not produce acetates and males are repelled by them. We previously found intraspecific variation in acetates in H. subflexa: females in eastern North America contain significantly more acetates than females in Western Mexico. Here we describe the persistence of this intraspecific variation in laboratory-reared strains and the identification of one major quantitative trait locus (QTL), explaining 40% of the variance in acetate amounts. We homologized this intraspecific QTL to our previously identified interspecific QTL using restriction-associated DNA (RAD) tags. We found that a major intraspecific QTL overlaps with one of the two major interspecific QTL. To identify candidate genes underlying the acetate variation, we investigated a number of gene families with known or suspected acetyl- or acyltransferase activity. The most likely candidate genes did not map to our QTL, so that we currently hypothesize that a transcription factor underlies this QTL. Finding a single, large QTL that impacts variation in pheromone blends between and within species is, to our knowledge, the first such example for traits that have been demonstrated to affect premating isolation.

Responses of Amblyomma americanum and Dermacentor variabilis to odorants that attract haematophagous insects.

Carbon dioxide (CO(2) ), 1-octen-3-ol, acetone, ammonium hydroxide, L-lactic-acid, dimethyl trisulphide and isobutyric acid were tested as attractants for two tick species, Amblyomma americanum and Dermacentor variabilis (Acari: Ixodidae), in dose-response bioassays using Y-tube olfactometers. Only CO(2) , acetone, 1-octen-3-ol and ammonium hydroxide elicited significant preferences from adult A. americanum, and only CO(2) was attractive to adult D. variabilis. Acetone, 1-octen-3-ol and ammonium hydroxide were separately evaluated at three doses against CO(2) (from dry ice) at a field site supporting a natural population of A. americanum nymphs and adults. Carbon dioxide consistently attracted the highest number of host-seeking ticks. However, for the first time, acetone, 1-octen-3-ol and ammonium hydroxide were shown to attract high numbers of A. americanum. Further research is needed to determine the utility of these semiochemicals as attractants in tick surveillance and area-wide management programmes.

Genetic differentiation across North America in the generalist moth Heliothis virescens and the specialist H. subflexa.

The two moth species Heliothis virescens (Hv) and H. subflexa (Hs) are closely related, but have vastly different feeding habits. Hv is a generalist and an important pest in many crops in the USA, while Hs is a specialist feeding only on plants in the genus Physalis. In this study, we conducted a comparative population genetic analysis to assess whether and how generalist and specialist life styles are reflected in differences in population structures. In Hv 98% of the total variation occurred within populations. The overall differentiation (F(ST) ) between regions was 0.006 and even lower between years (0.0039) and hosts (0.0028). Analyses of population structure suggest that all individuals form one genetically homogeneous population, except for at most 12 individuals (6%) that diverged from this cluster. Population homogeneity likely results from the high mobility of Hv and its generalist feeding behaviour. Hs exhibited substantially more population structure. Even though 96% of the total variation was attributable to within-population variability, F(ST) -values between Hs populations were 10 times higher than between Hv populations. Hs populations showed significant isolation by distance. Analyses of Hs population structure suggest at least two subpopulations and thus some degree of metapopulation structure. We speculate that the patchy distribution of Physalis- the exclusive food source of Hs - contributes to differences in population structure between these closely related species. The finding that the specialist shows more population differentiation than the generalist corroborates the notion that host specialization is not an evolutionary dead end but a dynamic trait.

Functional characterization of pheromone receptors in the tobacco budworm Heliothis virescens.

Functional analyses of candidate Heliothis virescens pheromone odorant receptors (HvORs) were conducted using heterologous expression in Xenopus oocytes. HvOR6 was found to be highly tuned to Z9-14:Ald, while HvOR13, HvOR14 and HvOR16 showed specificity for Z11-16:Ald, Z11-16:OAc and Z11-16:OH, respectively. HvOR15, which had been considered a candidate receptor for Z9-14:Ald did not respond to any of the pheromone compounds tested, nor to 50 other general odorants. Thus, while HvOR15 is specifically expressed in H. virescens male antennae, its role in pheromone reception remains unknown. Based on our results and previous research we can now assign pheromone receptors in H. virescens males to each of the critical H. virescens agonistic pheromone compounds and two antagonistic compounds produced by heterospecific females.

Phenotypic plasticity in sexual communication signal of a noctuid moth.

Variability within sex pheromone signalling systems is generally believed to be low because of strong stabilizing selection; yet the noctuid moth Heliothis subflexa (Hs) shows significant intraspecific variation. One possible explanation is that females may alter their sex pheromone blend depending on prevailing olfactory cues in the habitat, which we termed the 'experience hypothesis'. This could be adaptive if Hs females experiencing the pheromone of another species, Heliothis virescens (Hv), responded to reduce the frequency of heterospecific matings. We exposed Hs females to no pheromone, Hs pheromone or Hv pheromone in the first 3 days of their adult lives. Hs females in the latter treatment produced significantly more of the acetate Z11-16:OAc, which inhibits the attraction of Hv males. To our knowledge, this is the first study showing adaptive phenotypic plasticity in a moth sex pheromone and suggests that behavioural differentiation may precede genetic divergence in the sexual signals of moths.

Acoustic indicators for targeted detection of stored product and urban insect pests by inexpensive infrared, acoustic, and vibrational detection of movement.

Crawling and scraping activity of three stored-product pests, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), and Stegobium paniceum (L.) (Coleoptera: Anobiidae), and two urban pests, Blattella germanica (L.) (Blattodea: Blattellidae) and Cimex lectularius L. (Hemiptera: Cimicidae), were monitored individually by infrared sensors, microphones, and a piezoelectric sensor in a small arena to evaluate effects of insect locomotory behavior and size on the ability of an inexpensively constructed instrument to detect insects and distinguish among different species. Adults of all species could be detected when crawling or scraping. The smallest insects in the study, first-fourth-instar C. lectularius nymphs, could not be detected easily when crawling, but could be detected when scraping. Sound and vibration sensors detected brief, 3-10-ms impulses from all tested species, often grouped in distinctive trains (bursts), typical of impulses in previous acoustic detection experiments. To consider the potential for targeting or focusing detection on particular species of interest, indicators were developed to assess the likelihood of detection of C. lectularius. Statistically significant differences were found between C. lectularius and other species in distributions of three measured variables: infrared signal durations, sound impulse-burst durations, and sound pressure levels (energy) of impulses that best matched an averaged spectrum (profile) of scraping behavior. Thus, there is potential that signals collected by an inexpensive, polymodal-sensor instrument could be used in automated trapping systems to detect a targeted species, 0.1 mg or larger, in environments where servicing of traps is difficult or when timeliness of trapping information is important.

RNA interference-mediated knock-down of Bla g 1 in the German cockroach, Blattella germanica L., implicates this allergen-encoding gene in digestion and nutrient absorption.

We used RNA interference (RNAi) to silence the expression of a gene encoding Bla g 1, a human allergen produced by the German cockroach, Blattella germanica L., to study its function in cockroach physiology. Females injected with 1 microg of double-stranded RNA contained 64% less Bla g 1 protein and Bla g 1 mRNA abundance was reduced by 91.4% compared to sham-injected females. Bla g 1 knockdown slowed the pace of weight gain, midgut growth, and colleterial gland and basal oocyte maturation, resulting in delayed egg case formation and lower fecundity. Exogenous juvenile hormone treatments rescued reproduction in RNAi-treated females, suggesting that Bla g 1 silencing lowered endogenous juvenile hormone, probably by reducing food intake and nutrient absorption.

Intraspecific vertical stratification as a mate-finding mechanism in tropical cockroaches.

Cockroaches in a tropical forest stratify vertically both inter- and intraspecifically along micrometeorological gradients. At night, low wind speeds and unstable atmospheric conditions result in efficient vertical mixing of the air near the ground. Convective ascent of warm air imparts directionality to the pheromone- dispersion process. The occurrence of males at greater heights than pheromone- emitting conspecific females appears to be a mate-finding strategy.

Ecological correlates of paternal investment of urates in a tropical cockroach.

Females of the tropical cockroach Xestoblatta hamata feed on urates offered by the male after copulation. Females on nitrogen-deficient diets ingest and transfer to their maturing oocytes more male-derived uric acid than do females on high-protein diets. In isolated females, the greatest uptake of uric acid by the ovaries occurs during the mating stage in the reproductive cycle. Uric acid from males contributes significantly to the female's nitrogen pool and may help shorten the time between mating and oviposition. In both field and laboratory experiments males choose high-protein foods and dietary uric acid.

[Length polymorphism of integrated copies of R1 and R2 retrotransposons in the German cockroach (Blattella germanica) as a potential marker for population and phylogenetic studies].

Using polymerase chain reaction technique with primers flanking target sites of retrotransposons R1 and R2, integrated copies of these transposable elements were amplified in various cockroach species (Blattodea). It was shown that each species has a unique pattern of "5'-undertranscripts" with the definite set of amplified fragments of different lengths. Intraspecies polymorphism was revealed in analysis of German cockroach specimens obtained upon individual mating. This is the first report providing results of identifying, cloning, and sequencing extended fragments (5'-truncated copies) of Blatella germanica R1 and R2 retrotransposons. It may be assumed that patterns of 5'-truncated copies of R1 and R2 elements can be used as markers in population and phylogenetic studies. Moreover, cloned and sequenced fragments will be employed in our further studies for screening of the German cockroach genomic library in order to detect full-length copies in this class transposable elements.

[Nuclear rDNA variability in laboratory strains of the German cockroach Blattella germanica L. (Blattellidae)].

The polymorphism of nuclear ribosomal DNA has been studied in male German cockroaches. The RFLP analysis has been used to identify seven types of HindIII fragments, the variation of which may serve as the basis for the differentiation of laboratory strains with respect to some population indices, including the average number of fragments per animal (N), the proportion of polymorphic loci (P), and the average pairwise similarity (APS). The interpopulation variation (FST) calculated for nine haplotypes is 53.85%.

Neural and hormonal regulation of growth of corpora allata in the cockroach, Diploptera punctata.

DNA synthesis and mitosis in the corpora allata (CA) of adult Diploptera punctata males were investigated with total cell count after 5'-bromo-2'-deoxyuridine immunodetection and colchicine arrestment both in vivo and in vitro. The CA exhibited a single wave of DNA synthesis followed by cell division during the first 4 days after the imaginal ecdysis. A second mitotic wave was experimentally induced after the nervous connections between the CA and the brain were severed on day 4. Spontaneous mitosis was abolished in cockroaches treated with a juvenile hormone (JH) analog. This inhibitory regulation in vivo appeared to act through brain neurosecretory cells since in the denervated CA mitotic activity was unaffected by JH treatment. An in vitro system supporting growth of the corpus allatum was established to study direct hormonal effects. By using continuous bromodeoxyuridine labeling in vitro for 6 days, we showed that DNA synthesis of corpus allatum cells was unaffected by direct contact with JH. In contrast, 20-hydroxyecdysone exerted direct mitogenic action on allatal cells. These and previous results suggest that CA cells alternate between JH synthesis and a proliferative state in which they divide in a self-renewing fashion to yield differentiated progeny. We propose that in newly enclosed adult Diploptera punctata males, low JH titer and high ecdysteroid titer promote mitosis in CA cells. As the ecdysteroid titer declines, JH produced by the CA acts on brain neurosecretory cells which dispatch inhibitory signals through nerves to prevent continuous proliferation of CA cells.

Morphometric analysis of corpus allatum cells in adult females of three cockroach species.

The number of cells and their sizes in the corpus allatum (CA) of adult female Blattella germanica, Supella longipalpa and Diploptera punctata were determined during oocyte maturation. Cell number and size were directly measured in cell suspensions following enzymatic dissociation of freshly excised CA. Cell numbers were verified by total cell counts in whole-mount CA monolayers and by hemocytometric sampling. In all three species, cell number did not change during the period of CA activation, averaging ca. 2000 cells per gland in B. germanica, 3500 cells per gland in S. longipalpa and 11,000 cells per gland in D. punctata. Cell diameter increased significantly in all three species during this period from a mean value of 8.9 microns to 11.7 microns in B. germanica, from 9.2 microns to 14.6 microns in S. longipalpa and from 10.0 microns to 15.6 microns in D. punctata. During a 4 h incubation period, dissociated CA cells incorporated L-[methyl-3H]-methionine into juvenile hormone-III at rates comparable to intact glands. These data suggest that CA activation in the first ovarian cycle of these species is associated mainly with an increase in cell size with minor changes in cell number.

Developmental regulation of juvenile hormone synthesis: ovarian synchronization of volumetric changes of corpus allatum cells in cockroaches.

The corpus allatum (CA) cells of adult Blattella germanica females undergo cyclic volumetric changes in relation to juvenile hormone (JH) synthesis. In intact females the size of CA cells changes synchronously during the gonotrophic cycle, resulting in cyclic JH synthesis. In ovariectomized females volumetric changes among CA cells become asynchronous, resulting in highly variable but high rates of JH synthesis. Injection of the steroid hormone 20-hydroxyecdysone into ovariectomized females with active CA resulted in a transient decline followed by an increase in both CA volume and JH biosynthesis. This response was due to a change in the size distribution of CA cells and not in the total number of CA cells. In ovariectomized females, CA cells can be re-synchronized into a uniform population of small inactive cells with injection of 20-hydroxyecdysone and implantation of an artificial egg-case, mimicking the successive events of ovulation, oviposition and pregnancy.