Coby Schal

Identification of bacteria and bacteria-associated chemical cues that mediate oviposition site preferences by Aedes aegypti

The yellow fever mosquito, Aedes aegypti, the global vector of dengue and yellow fever, is inexorably linked to water-filled human-made containers for egg laying and production of progeny. Oviposition is stimulated by cues from water containers, but the nature and origin of these cues have not been elucidated. We showed that mosquito females directed most of their eggs to bamboo and white-oak leaf infusions, and only a small fraction of the eggs were laid in plain water containers. In binary choice assays, we demonstrated that microorganisms in leaf infusions produced oviposition-stimulating kairomones, and using a combination of bacterial culturing approaches, bioassay-guided fractionation of bacterial extracts, and chemical analyses, we now demonstrate that specific bacteria-associated carboxylic acids and methyl esters serve as potent oviposition stimulants for gravid Ae. aegypti. Elucidation of these compounds will improve understanding of the chemical basis of egg laying behavior of Ae. aegypti, and the kairomones will likely enhance the efficacy of surveillance and control programs for this disease vector of substantial global public health importance.

Diversity and contribution of the intestinal bacterial community to the development of Musca domestica (Diptera: Muscidae) larvae

Abstract The bacterial diversity in the intestinal tract of Musca domestica L. was examined in larvae collected from turkey bedding and corn silage. Aerobic culturing yielded 25 bacterial species, including 11 from larvae collected from turkey bedding and 14 from larvae collected from corn silage. Providencia rettgeri (Hadley, Elkins & Caldwell) was the only species common to both environments. Two mammalian pathogens, Yersinia pseudotuberculosis (Pfeiffer) and Ochrobactrum anthropi (Holmes), were isolated from the larval intestinal tracts. The majority of isolates represented facultatively anaerobic heterotrophs capable of fermentation. The significance of these bacteria for development of house fly larvae was evaluated by bioassays on trypticase soy egg yolk agar. Pure cultures of individual bacterial species isolated from the intestinal tract of larvae from turkey bedding supported development of flies to a much greater extent than those isolated from larvae from corn silage. House fly development was best supported by a Streptococcus sanguis (White) isolate. The significance of bacteria for development of house flies is discussed.

Pheromone puff trajectory and upwind flight of male gypsy moths in a forest

ABSTRACT. Pheromone released from a point source beneath a forest canopy usually follows a non‐linear trajectory as demonstrated by the paths of small, neutrally‐buoyant, helium‐filled balloons or puffs of smoke. Mark‐recapture experiments show that few male gypsy moths (Lymantria dispar L.) follow a pheromone plume over distances greater than 80 m even though they can easily detect pheromone at that distance as indicated by wingfanning assay. The directional consistency of successive puffs of pheromone appears more important than the linearity of their trajectories in enabling males to locate a pheromone source.

BEHAVIOURAL ECOLOGY OF COCKROACHES

1. Cockroaches are ubiquitous in most habitats where insects occur. Although most reports on cockroaches are physiological in nature, sufficient information is available to indicate that forest, desert, and cave‐dwelling cockroaches select microhabitats on the basis of finely resolved environmental preferences. This is particularly true for oviparous females which select specific substrates for oviposition and embryogenesis. Selection and diel movements between microhabitats are related to diel changes in micrometeorological profiles and predation, feeding, and enhancement of sexual communication.

Reproductive biology of the German cockroach, Blattella germanica: Juvenile hormone as a pleiotropic master regulator

Juvenile hormone (JH) exerts major pleiotropic effects on cockroach development and reproduction. The production of JH by the corpora allata (CA) in the adult female German cockroach, Blattella germanica, is dependent upon and modulated by both internal and environmental stimuli. Mating, intake of highquality food, social interactions, and the presence of vitellogenic ovaries facilitate JH synthesis. Conversely, starvation, deficient diets, enforced virginity, isolation, and a pre- or post-vitellogenic ovary cause the CA to produce less JH. Sensory stimulation of the genital vestibulum by the ootheca also inhibits the CA via signals that ascend the ventral nerve cord. All these stimulatory and inhibitory signals are integrated by the brain, and a preponderance of favorable signals results in a graded lifting of brain inhibition, permitting the synthesis and release of JH. The effects of inhibitory signals on JH biosynthesis can be lifted experimentally by severing nervous connections between the brain and the CA. Such an operation accelerates activation of the CA. Besides controlling gonadal maturation in females, JH concurrently regulates the production of sexual signals, including both attractant- and courtship-eliciting pheromones, and the behavioral expression of calling (pheromone release) and sexual receptivity. Although JH is required for the expression of copulatory readiness in female B. germanica, it appears that signals associated with copulation (spermatophore, sperm, accessory secretions) can inhibit this behavioral state even when titers of JH are permissive for receptivity. These observations suggest that JH might regulate sexual receptivity in females indirectly through

RAPID EVOLUTION OF CUTICULAR HYDROCARBONS IN A SPECIES RADIATION OF ACOUSTICALLY DIVERSE HAWAIIAN CRICKETS (GRYLLIDAE: TRIGONIDIINAE: LAUPALA)

Abstract Understanding the origin and maintenance of barriers to gene exchange is a central goal of speciation research. Hawaiian swordtail crickets (genus Laupala) represent one of the most rapidly speciating animal groups yet identified. Extensive acoustic diversity, strong premating isolation, and female preference for conspecific acoustic signals in laboratory phonotaxis trials have strongly supported divergence in mate recognition as the driving force behind the explosive speciation seen in this system. However, recent work has shown that female preference for conspecific male calling song does not extend to mate choice at close range among these crickets, leading to the hypothesis that additional sexual signals are involved in mate recognition and premating isolation. Here we examine patterns of variation in cuticular lipids among several species of Laupala from Maui and the Big Island of Hawaii. Results demonstrate (1) a rapid and dramatic evolution of cuticular lipid composition among species in this genus, (2) significant differences among males and females in cuticular lipid composition, and (3) a significant reduction in the complexity of cuticular lipid profiles in species from the Big Island of Hawaii as compared to two outgroup species from Maui. These results suggest that behavioral barriers to gene exchange in Laupala may be composed of multiple mate recognition signals, a pattern common in other cricket species.

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.

Role of bacteria in mediating the oviposition responses of Aedes albopictus (Diptera: Culicidae).

Abstract The responses of Aedes albopictus to sources of oviposition attractants and stimulants were evaluated with a behavioral bioassay in which females attracted to odorants emanating from water were trapped on screens coated with an adhesive. Gravid mosquitoes were attracted to volatiles from larval-rearing water and soil-contaminated cotton towels. Bacteria were isolated from these substrates and from an organic infusion made with oak leaves. Through fatty acid-methyl ester analyses, six bacterial isolates from larval-rearing water, two isolates from soil-contaminated cotton towels, and three isolates from oak leaf infusion were identified to species. The response of gravid mosquitoes to these isolates was also evaluated in behavioral bioassays. Water containing Psychrobacter immobilis (from larval-rearing water), Sphingobacterium multivorum (from soil-contaminated cotton towels), and an undetermined Bacillus species (from oak leaf infusion) elicited significantly higher oviposition than control water without bacteria. Only volatiles collected from larval rearing water elicited significant electroantennogram responses in females.

Endocrine regulation of de novo aggregation pheromone biosynthesis in the pine engraver, Ips pini (Say) (Coleoptera: Scolytidae)

Abstract In vivo and in vitro radiotracer studies were conducted with the pine engraver, Ips pini (Say) (Coleoptera: Scolytidae), to elucidate the relationships among feeding on host ( Pinus jeffreyi Grev. & Balf.) phloem, juvenile hormone III (JH III) biosynthesis, and de novo aggregation pheromone (ipsdienol) biosynthesis. The in vivo incorporation of [1- 14 C]acetate into ipsdienol by male I. pini increased with increasing dose of topically-applied JH III, demonstrating the stimulatory role by JH III in de novo pheromone production. In vivo incorporation of ( RS )-[2- 14 C]mevalonolactone into ipsdienol by male I. pini was not affected by increasing JH III dose. However, injection of [ 14 C]mevalonolactone resulted in significantly higher levels of [ 14 C]ipsdienol than those observed in saline-injected controls. This is direct evidence for the mevalonate-based isoprenoid pathway in de novo ipsdienol biosynthesis, and suggests that in this pathway JH III does not influence enzymatically-catalyzed reactions subsequent to the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonate. An additional in vivo [ 14 C]acetate study demonstrated that de novo ipsdienol biosynthesis is also stimulated by feeding on host phloem. Lastly, an in vitro radiotracer study utilizing L-[ methyl - 3 H]methionine demonstrated that feeding stimulates JH III biosynthesis by the corpora allata (CA) of male, but not female, I. pini . Analysis by radio-high pressure liquid chromatography revealed that JH III is likely the type of juvenile hormone produced by the male CA. These data support a sequence of events leading to feeding-induced de novo pheromone biosynthesis in male I. pini : (1) feeding on host phloem; (2) feeding-dependent JH III biosynthesis by the CA; and (3) JH III-stimulated de novo ipsdienol biosynthesis.

Insects in confined swine operations carry a large antibiotic resistant and potentially virulent enterococcal community

BackgroundExtensive use of antibiotics as growth promoters in the livestock industry constitutes strong selection pressure for evolution and selection of antibiotic resistant bacterial strains. Unfortunately, the microbial ecology and spread of these bacteria in the agricultural, urban, and suburban environments are poorly understood. Insects such as house flies (Musca domestica) and German cockroaches (Blattella germanica) can move freely between animal waste and food and may play a significant role in the dissemination of antibiotic resistant bacteria within and between animal production farms and from farms to residential settings.ResultsEnterococci from the digestive tract of house flies (n = 162), and feces of German cockroaches (n = 83) and pigs (n = 119), collected from two commercial swine farms were isolated, quantified, identified, and screened for antibiotic resistance and virulence. The majority of samples (93.7%) were positive for enterococci with concentrations 4.2 ± 0.7 × 104 CFU/house fly, 5.5 ± 1.1 × 106 CFU/g of cockroach feces, and 3.2 ± 0.8 × 105 CFU/g of pig feces. Among all the identified isolates (n = 639) Enterococcus faecalis was the most common (55.5%), followed by E. hirae (24.9%), E. faecium (12.8%), and E. casseliflavus (6.7%). E. faecalis was most prevalent in house flies and cockroaches, and E. hirae was most common in pig feces. Our data showed that multi-drug (mainly tetracycline and erythromycin) resistant enterococci were common from all three sources and frequently carried antibiotic resistance genes including tet(M) and erm(B) and Tn916/1545 transposon family. E. faecalis frequently harbored virulence factors gelE, esp, and asa1. PFGE analysis of selected E. faecalis and E. faecium isolates demonstrated that cockroaches and house flies shared some of the same enterococcal clones that were detected in the swine manure indicating that insects acquired enterococci from swine manure.ConclusionsThis study shows that house flies and German cockroaches in the confined swine production environment likely serve as vectors and/or reservoirs of antibiotic resistant and potentially virulent enterococci and consequently may play an important role in animal and public health.