Evaluation of Commercial 6-Phytases on Growth Performance, Bone Mineral Content, and Feed Digestibility of Broiler Chicks.

We evaluated the effects of 6-phytases on the growth performance of broilers (UK Chunky) under the recommended supplier-application dosage of each phytase. A nutritionally sufficient standard diet was administered as the base diets in the positive control feed. The diet in the negative control feed was designed by reducing total phosphorous, non-phytate phosphorus, and calcium by 0.1% to evaluate the effect of the nutrient restriction on broilers. Four 6-phytases were added to negative control feeds at the level of the recommended dosage of each product to compare the effect of phytases on broiler technical performance, tibia ash, and feed digestibility. Nine hundred one-day-old broiler chicks (males and females) were distributed in a completely randomized design composed of six treatments and three replicates of 50 chicks each. Chicks were fed ad libitum for 49 days. Body weight gain and feed intake were recorded on days 21 and 49, tibia ash was measured on day 21, and apparent ileal digestibility of dry matter, crude protein, and total phosphorus were analyzed on day 49. Birds reared with test feeds supplemented with phytase showed higher body weight gain and feed intake compared to those of the negative control birds. No significant differences in traits were observed among different phytase treatments. Similarly, the percentage of tibia ash increased when phytase was supplemented, resulting in higher bone levels compared to that of the positive control. The apparent ileal digestibility of crude protein and total phosphorus was enhanced by supplementing negative control diets with phytases.

Control of Fusarium and nematodes by entomopathogenic fungi for organic production of Zingiber officinale.

Ginger (genus Zingiber) is widely used as a spice and a medicinal herb worldwide and is the major ingredient of traditional local drinks such as jamu in Southeast Asia. Because ginger is frequently consumed, there is an increasing interest in organic ginger production without the use of synthetic agrochemicals. Recent studies have reported that certain kinds of entomopathogenic fungi (EPF) can establish endophytic- or mycorrhiza-like relationships with plants, thereby promoting plant growth and health, in addition to their typical role in crop protection as biological control agents. In this study, we explored the possibility of non-entomopathogenic effects of EPF Beauveria bassiana and Cordyceps fumosorosea on ginger plants (Zingiber officinale) via antagonism with Fusarium oxysporum or the parasitic nematode Meloidogyne incognita. The two EPF negatively affected the growth of F. oxysporum and survival of M. incognita in vitro. The application of EPF did not have any negative effect on the growth of ginger plants. Soil chemical properties were not different between the plots with or without EPF application, while the diversity of soil bacteria was observed to increase on application of EPF. At least C. fumosorosea appeared to persist in soil during the period of ginger cultivation. Thus, these EPF are potentially useful tools for producing chemical-free ginger.

Impact of 2.45 GHz Microwave Irradiation on the Fruit Fly, Drosophila melanogaster.

The physiological and behavioral influences of 2.45 GHz microwaves on Drosophila melanogaster were examined. Standing waves transitioned into heat energy effectively when passing through the insect body. On the contrary, travelling waves did not transit into heat energy in the insect body. This indicated that there was no concern regarding the thermal effects of microwave irradiation for levels of daily usage. However, we detected genotoxicity and behavioral alterations associated with travelling wave irradiation, which can be attributed to the non-thermal effects of the waves. Electron spin resonance (ESR) revealed that fruit flies possessed paramagnetic substances in the body such as Fe3+, Cu2+, Mn2+, and organic radicals. The temperature dependent intensities of these paramagnetic substances indicated that females possessed more of the components susceptible to electromagnetic waves than males, and the behavioral tests supported the differences between the sexes.

Genetic Basis of Natural Variation in Spontaneous Grooming in Drosophila melanogaster.

Spontaneous grooming behavior is a component of insect fitness. We quantified spontaneous grooming behavior in 201 sequenced lines of the Drosophila melanogaster Genetic Reference Panel and observed significant genetic variation in spontaneous grooming, with broad-sense heritabilities of 0.25 and 0.24 in females and males, respectively. Although grooming behavior is highly correlated between males and females, we observed significant sex by genotype interactions, indicating that the genetic basis of spontaneous grooming is partially distinct in the two sexes. We performed genome-wide association analyses of grooming behavior, and mapped 107 molecular polymorphisms associated with spontaneous grooming behavior, of which 73 were in or near 70 genes and 34 were over 1 kilobase from the nearest gene. The candidate genes were associated with a wide variety of gene ontology terms, and several of the candidate genes were significantly enriched in a genetic interaction network. We performed functional assessments of 29 candidate genes using RNA interference, and found that 11 affected spontaneous grooming behavior. The genes associated with natural variation in Drosophila grooming are involved with glutamate metabolism (Gdh) and transport (Eaat); interact genetically with (CCKLR-17D1) or are in the same gene family as (PGRP-LA) genes previously implicated in grooming behavior; are involved in the development of the nervous system and other tissues; or regulate the Notch and Epidermal growth factor receptor signaling pathways. Several DGRP lines exhibited extreme grooming behavior. Excessive grooming behavior can serve as a model for repetitive behaviors diagnostic of several human neuropsychiatric diseases.

Physical assessments of termites (Termitidae) under 2.45 GHz microwave irradiation.

Demands for chemical-free treatments for controlling insect pests are increasing worldwide. One such treatment is microwave heating; however, two critical issues arise when using microwaves as a heat source: intensive labor and excessive energy-consumption. Optimization is thus required to reduce energy consumption while effectively killing insects. Currently, the lethal effect of microwaves on insects is considered to be due to the temperature of the irradiated materials. This study examines how the conditions of irradiation, such as resonance or traveling mode, changed the conversion of electromagnetic energy into heat when 2.45 GHz microwaves penetrated the body of the termite, C. formosanus. Our results indicated that it is possible to heat and kill termites with microwaves under resonance condition. Termites were however found to be very tolerant to microwave irradiation as the permittivity of the insect was low compared with other reported insects and plants. Electron spin resonance revealed that termites contained several paramagnetic substances in their bodies, such as Fe3+, Cu2+, Mn2+, and organic radicals. Interestingly, irradiation with traveling microwaves hardly produced heat, but increased the organic radicals in termite bodies indicating non-thermal effects of microwaves.

LPS perception through taste-induced reflex in Drosophila melanogaster.

In flies, grooming serves several purposes, including protection against pathogens and parasites. Previously, we found Escherichia coli or lipopolysaccharides (LPS) can induce grooming behavior via activation of contact chemoreceptors on Drosophila wing. This suggested that specific taste receptors may contribute to this detection. In this study, we examined the perception of commercially available LPS on Drosophila wing chemoreceptors in grooming reflex. Behavioral tests conducted with bitter, sweet and salty gustation such as caffeine, sucrose and salt, using flies carrying a defect in one of their taste receptors related to the detection of bitter molecules (Gr66a, Gr33a), sugars (Gr5a, Gr64f), or salt (IR76b). LPS and tastants of each category were applied to wing sensilla of these taste defectflies and to wild-type Canton Special (CS) flies. Our results indicate that the grooming reflex induced by LPS requires a wide range of gustatory genes, and the inactivation of any of tested genes expressing cells causes a significant reduction of the behavior. This suggests that, while the grooming reflex is strongly regulated by cues perceived as aversive, other sapid cues traditionally related to sweet and salty tastes are also contributing to this behavior.

Soy undecapeptide induces Drosophila hind leg grooming via dopamine receptor.

ß-Conglycinin α subunit (323-333) [ßCGα(323-333)] is an exogenous neuromodulating undecapeptide found from enzymatic digest of ß-conglycinin, a soy major storage protein by mice behavior tests. We investigated effect of ßCGα(323-333) on Drosophila behavior. Oral administration of ßCGα(323-333) in Drosophila increased hind leg grooming, which may act through specific sets of neurons. It was reported that dopamine receptor (DopR) meditates hind leg grooming, and we tested involvement of DopR in ßCGα(323-333)-induced hind leg grooming by using DopR knockout flies. In the wild type but not in the DopR-knockout flies, ßCGα(323-333) increased hind leg grooming. These results suggest that ßCGα(323-333) induces hind leg grooming via activating the DopR. This is the first report showing that exogenously administered peptide changes fly behaviors.

Olfactory cues play a significant role in removing fungus from the body surface of Drosophila melanogaster.

Many insects and Dipterans in particular are known to spend considerable time grooming, but whether these behaviors actually are able to remove pathogenic fungal conidia is less clear. In this study, we examined whether grooming serves to protect flies by reducing the risk of fungal infection in Drosophila melanogaster. First, we confirmed that fungi were removed by grooming. Entomopathogenic, opportunistic, and plant pathogenic fungi were applied on the body surface of the flies. To estimate grooming efficiency, the number of removal conidia through grooming was quantified and we successfully demonstrated that flies remove fungal conidia from their body surfaces via grooming behavior. Second, the roles of gustatory and olfactory signals in fungus removal were examined. The wildtype fly Canton-S, the taste deficiency mutant poxn 70, and the olfactory deficiency mutant orco1 were used in the tests. Comparisons between Canton-S and poxn 70 flies indicated that gustatory signals do not have a significant role in fungal removal via grooming behavior in D. melanogaster. In contrast, the efficiency of conidia removal in orco1 flies was drastically decreased. Consequently, this study indicated that flies rely on mechanical stimulus for the induction of grooming and olfaction for more detailed removal.

The gram-negative sensing receptor PGRP-LC contributes to grooming induction in Drosophila.

Behavioral resistance protects insects from microbial infection. However, signals inducing insect hygiene behavior are still relatively unexplored. Our previous study demonstrated that olfactory signals from microbes enhance insect hygiene behavior, and gustatory signals even induce the behavior. In this paper, we postulated a cross-talk between behavioral resistance and innate immunity. To examine this hypothesis, we employed a previously validated behavioral test to examine the function of taste signals in inducing a grooming reflex in decapitated flies. Microbes, which activate different pattern recognition systems upstream of immune pathways, were applied to see if there was any correlation between microbial perception and grooming reflex. To narrow down candidate elicitors, the grooming induction tests were conducted with highly purified bacterial components. Lastly, the role of DAP-type peptidoglycan in grooming induction was confirmed. Our results demonstrate that cleaning behavior can be triggered through recognition of DAP-type PGN by its receptor PGRP-LC.

Seawater influence monitored by NaCl on the growth of Trametes versicolor.

There are only a few scientific data about the function of ecosystems after tsunami disasters. The ecosystems help the environment to recover after a disaster, and therefore, the research on its function is important. We estimated the seawater influences on wood degradation after a tsunami disaster by the growth of Trametes versicolor. The debris from the Great East Japan Earthquake on the pacific coast in March 2011 was used for the simulations. Its growth on debris was compared with those on seawater-treated woods, and the amount of sodium chloride was examined to know the approximate amount of salts in the samples. Sodium chloride contents were employed as an indicative parameter of sea salts, which contained many elements. As a result, this common white-rot fungus degraded wood debris in the same way as sound sapwood. Although the study was conducted at the laboratory level, this is the first report from the real debris, which assessed the fungal decomposition ability of the ecosystem after a tsunami disaster.

Drosophila Bitter Taste(s).

Most animals possess taste receptors neurons detecting potentially noxious compounds. In humans, the ligands which activate these neurons define a sensory space called "bitter". By extension, this term has been used in animals and insects to define molecules which induce aversive responses. In this review, based on our observations carried out in Drosophila, we examine how bitter compounds are detected and if bitter-sensitive neurons respond only to molecules bitter to humans. Like most animals, flies detect bitter chemicals through a specific population of taste neurons, distinct from those responding to sugars or to other modalities. Activating bitter-sensitive taste neurons induces aversive reactions and inhibits feeding. Bitter molecules also contribute to the suppression of sugar-neuron responses and can lead to a complete inhibition of the responses to sugar at the periphery. Since some bitter molecules activate bitter-sensitive neurons and some inhibit sugar detection, bitter molecules are represented by two sensory spaces which are only partially congruent. In addition to molecules which impact feeding, we recently discovered that the activation of bitter-sensitive neurons also induces grooming. Bitter-sensitive neurons of the wings and of the legs can sense chemicals from the gram negative bacteria, Escherichia coli, thus adding another biological function to these receptors. Bitter-sensitive neurons of the proboscis also respond to the inhibitory pheromone, 7-tricosene. Activating these neurons by bitter molecules in the context of sexual encounter inhibits courting and sexual reproduction, while activating these neurons with 7-tricosene in a feeding context will inhibit feeding. The picture that emerges from these observations is that the taste system is composed of detectors which monitor different "categories" of ligands, which facilitate or inhibit behaviors depending on the context (feeding, sexual reproduction, hygienic behavior), thus considerably extending the initial definition of "bitter" tasting.

Olfactory Cues from Pathogenic Fungus Affect the Direction of Motion of Termites, Coptotermes formosanus.

Formosan subterranean termites, Coptotermes formosanus, tend to avoid pathogen odors when tested in Y-tube olfactometers, but approach and groom exposed nestmates to remove pathogens from their cuticle and maintain a healthy population. To better understand their differential reaction to pathogens and their odors, the relationship between odor cues and direction of motion was examined with the fungus Isaria fumosorosea K3 strain. The results indicate that nestmate odor was strongly attractive only in tests where fungal odors were present in both branches of the olfactometer. Termites generally avoid fungal odors when offered a choice without fungal odor. We also tested termite aversion to 3-octanone and 1-octen-3-ol, major surface chemical compounds of I. fumosorosea K3, and estimated the total mass of these compounds present on the conidial surface by direct extraction method. The total quantity of these chemicals on the surface of fungal conidia was estimated to be approximately 0.01 ng per 10(7) conidia. This study demonstrates a context dependent behavioral change in termites in response to the odors of pathogenic fungi.

Hygienic grooming is induced by contact chemicals in Drosophila melanogaster.

In social insects, grooming is considered as a behavioral defense against pathogen and parasite infections since it contributes to remove microbes from their cuticle. However, stimuli which trigger this behavior are not well characterized yet. We examined if activating contact chemoreceptive sensilla could trigger grooming activities in Drosophila melanogaster. We monitored the grooming responses of decapitated flies to compounds known to activate the immune system, e.g., dead Escherichia coli (Ec) and lipopolysaccharides (LPS), and to tastants such as quinine, sucrose, and salt. LPS, quinine, and Ec were quite effective in triggering grooming movements when touching the distal border of the wings and the legs, while sucrose had no effect. Contact chemoreceptors are necessary and sufficient to elicit such responses, as grooming could not be elicited by LPS in poxn mutants deprived of external taste sensilla, and as grooming was elicited by light when a channel rhodopsin receptor was expressed in bitter-sensitive cells expressing Gr33a. Contact chemoreceptors distributed along the distal border of the wings respond to these tastants by an increased spiking activity, in response to quinine, Ec, LPS, sucrose, and KCl. These results demonstrate for the first time that bacterial compounds trigger grooming activities in D. melanogaster, and indicate that contact chemoreceptors located on the wings participate in the detection of such chemicals.

Grooming Behavior as a Mechanism of Insect Disease Defense.

Grooming is a well-recognized, multipurpose, behavior in arthropods and vertebrates. In this paper, we review the literature to highlight the physical function, neurophysiological mechanisms, and role that grooming plays in insect defense against pathogenic infection. The intricate relationships between the physical, neurological and immunological mechanisms of grooming are discussed to illustrate the importance of this behavior when examining the ecology of insect-pathogen interactions.

Odor aversion and pathogen-removal efficiency in grooming behavior of the termite Coptotermes formosanus.

The results of biocontrol with entomopathogens in termites have been discouraging because of the strong social hygiene behavior for removing pathogens from termite colonies. However, the mechanism of pathogen detection is still unclear. For the successful application of biopesticides to termites in nature, it would be beneficial to identify substances that could disrupt the termite's ability to perceive pathogens. We hypothesized that termites can perceive pathogens and this ability plays an important role in effective hygiene behavior. In this study, pathogen-detection in the subterranean termite Coptotermes formosanus was investigated. We performed quantitative assays on conidia removal by grooming behavior using epifluoresence microscopy and Y-maze tests to examine the perception of fungal odor by termites. Three species each of high- and low-virulence entomopathogenic fungi were used in each test. The results demonstrated that termites removed conidia more effectively from a nestmate's cuticle if its odor elicited stronger aversion. Highly virulent pathogens showed higher attachment rates to termite surfaces and their odors were more strongly avoided than those of low-virulence isolates in the same species. Moreover, termites appeared to groom each other more persistently when they had more conidia on their bodies. In brief, insect perception of pathogen-related odor seems to play a role in the mechanism of their hygiene behavior.

Musty odor of entomopathogens enhances disease-prevention behaviors in the termite Coptotermes formosanus.

Termites often eliminate pathogens directly through mutual grooming, and are thereby prevent infections from entomopathogenic fungi. Our previous study confirmed that the antennae of Coptotermesformosanus sensitively responded to the musty odor of entomopathogenic fungi. However, it is unclear if this odor has any effect on termite behavior. The purpose of this study was to clarify the effects of fungal odor on termite behavior, especially on conidia removal. The musty odor was prepared as an aqueous solution by immersing conidia in distilled water. When untreated termites were mixed with fungal-odor-treated termites at a ratio of 4:1, mutual grooming and attack of treated termites were frequently observed. This indicated that the fungal odor triggered these behavioral responses. While some components of the fungal odor were found in all of the entomopathogenic fungi tested, the odor profiles differed among the isolates.

Behavioral changes in the termite, Coptotermes formosanus (Isoptera), inoculated with six fungal isolates.

The studies of pathogen-prevention behaviors of termites have focused on hygiene behavior directed only against highly virulent pathogens. Therefore, we compared behavioral changes in the subterranean termite Coptotermes formosanus following contact with entomopathogenic fungi with different levels of virulence. The fungal virulence was inferred from the daily mortality and the LD50 value in previous data. When untreated termites were allowed to contact their fungus-inoculated nestmates, mutual grooming was frequent during 30 min after inoculation. The inoculated termites were often attacked and eaten by their uninoculated nestmates, and then buried after death. Notably, there was no influence of fungal virulence on these pathogen-prevention behaviors. However, the fungal isolates and genera affected not only the frequency of the behaviors but also the horizontal transmission pattern, the number of dead individuals and the survival period before the first death following infection.

Influence of fungal odor on grooming behavior of the termite, Coptotermes formosanus.

The termite Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) protects itself from entomopathogenic fungus by mutual grooming behavior. C. formosanus removes foreign organisms, such as fungal conidia, from the body surface of its nestmates by mutual grooming behavior and eating them. The conidia removal rate from the body surface differed according to the isolate of entomopathogenic fungi (Beauveria brongniartii 782, Paecilomyces fumosoroseus K3, and Metarhizium anisopliae 455), and the removal rate of the fungal isolates seemed to depend on feeding preference, which was determined using paper discs moistened with a fungal suspension. In addition, it was found that C. formosanus without antennae groomed their nestmates more frequently than those with antennae. Consequently, it seems that C. formosanus antennae detect substances without touching, such as via odor, and it affects the efficiency of grooming behavior. The results of single sensillum recording support the hypothesis that C. formosanus are capable of distinguishing three species of fungi by their odors.

The role of antennae in removing entomopathogenic fungi from cuticle of the termite, Coptotermes formosanus.

Our previous research has shown that the termite, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), protects itself from entomopathogenic fungi by mutual grooming behavior. The termite removes and discards foreign organisms, such as fungal conidia, from the body surface of its nestmates by mutual grooming behavior. The role of the antennae in detecting the condia was examind here. Three entomopathogenic fungi were used, Beauveria brongniartii 782 (Saccardo) (Hypocreales), Paecilomyces fumosoroseus K3 (Wize) (Hyphomycetes), and Metarhizium anisopliae 455 Sorokin (Hyphomycetes). Termites with antennae removed conidia more efficiently than termites without antennae. There were differences between termites with and without antennae in selection of sites to be groomed on nestmates, in the length of grooming and in occurrence of grooming. Electroantennogram (EAG) responses were recorded from termite antennae and the waveforms were rather specific to the kinds of fungi used as odor sources. Termites were able to distinguish between the tested fungi in feeding tests. These results show that the antennae play important roles in the mutual grooming behavior of the termite.