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Many parasitoids discriminate previously parasitised hosts from unparasitised ones to avoid mortality of offspring. Parasitoids that parasitise aggressive hosts such as lepidopteran larvae are known to attack hosts very quickly to avoid being attacked. However, little is known about host discrimination of such quick attacking parasitoids. We investigated host discrimination of Microplitis demolitor (Wilkinson) (Hymenoptera: Braconidae) a quick attacking parasitoid of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). Results showed that ratios of female wasps that rejected the hosts after antennal examination did not differ between parasitised and unparasitised hosts, indicating that M. demolitor did not discriminate hosts by antennal examination. However, 95% of females that inserted ovipositor into unparasitised hosts actually laid eggs, whereas it was only 31% for parasitised hosts, indicating that females discriminated hosts by oviposition insertion. Analyzing video recordings revealed that the ovipositor exploration of the host took 0.3 s. Female wasps that had experienced high-host density of unparasitised hosts readily rejected parasitised hosts, while wasps with experience of low host availability of parasitised hosts tended to accept parasitised hosts. This suggests that host discrimination behaviour of M. demolitor is affected by previous experience of different host availability and host quality.

So far, Phenacoccis manihoti (Hemiptera: Pseudococcidae) has been reared mainly with cassava. To explore alternative host plant for rearing of P. manihoti and its parasitoid Anagyrus lopezi De Santis (Hymenoptera: Encyrtidae), we examined suitability of taro Colocasia esculenta (L.) (Alismatales: Araceae) for rearing them in the laboratory, which was compared with that of cassava seedling. Either 50 newly hatched mealybug nymphs or parasitized third-instar mealybugs were reared on taro or cassava seedlings to examine immature development, fecundity and longevity of adults. Nearly 90% of P. manihoti reared on taro successfully developed to adults and they produced 230 eggs on average in their lifetime, while insects reared on cassava showed 95% survival and 250 eggs of lifetime fecundity. For A. lopezi, immature survival was 86% and they laid 140 eggs on average on taro, while it was 88% and 140 eggs on cassava. The results suggested that taro can be used for rearing of P. manihoti and its parasitoid A. lopezi.

The northern tree shrew (Tupaia belangeri) possesses high potential as an animal model of human diseases and biology, given its genetic similarity to primates. Although genetic information on the tree shrew has already been published, some of the entire coding sequences (CDSs) of tree shrew genes remained incomplete, and the reliability of these CDSs remained difficult to determine. To improve the determination of tree shrew CDSs, we performed sequencing of the whole-genome, mRNA, and total RNA and integrated the resulting data. Additionally, we established criteria for the selection of reliable CDSs and annotated these sequences by comparison to the human transcriptome, resulting in the identification of complete CDSs for 12,612 tree shrew genes and yielding a more accurate tree shrew genome database (TupaiaBase: http://tupaiabase.org). Transcriptome profiles in hepatitis B virus infected tree shrew livers were analyzed for validation. Gene ontology analysis showed enriched transcriptional regulation at 1 day post-infection, namely in the “type I interferon signaling pathway”. Moreover, a negative regulator of type I interferon, SOCS3, was induced. This work, which provides a tree shrew CDS database based on genomic DNA and RNA sequencing, is expected to serve as a powerful tool for further development of the tree shrew model.

Light is an indispensable part of routine laboratory work in which conventional light is generally used. Light-emitting diodes (LEDs) have come to replace conventional light, and thus could be a potent target in biomedical studies. Since blue light is a major component of visible light wavelength, in this study, using a somatic cell from the African green monkey kidney, we assessed the possible consequences of the blue spectra of LED light in future animal experiments and proposed a potent mitigation against light-induced damage. COS-7 cells were exposed to blue LED light (450 nm) and the growth and deoxyribonucleic acid (DNA) damage were assessed at different exposure times. A higher suppression in cell growth and viability was observed under a longer period of blue LED light exposure. The number of apoptotic cells increased as the light exposure time was prolonged. Reactive oxygen species (ROS) generation was also elevated in accordance to the extension of light exposure time. A comparison with dark-maintained cells revealed that the upregulation of ROS by blue LED light plays a significant role in causing cellular dysfunction in DNA in a time-dependent manner. In turn, antioxidant treatment has been shown to improve cell growth and viability under blue LED light conditions. This indicates that antioxidants have potential against blue LED light-induced somatic cell damage. It is expected that this study will contribute to the understanding of the basic mechanism of somatic cell death under visible light and maximize the beneficial use of LED light in future animal experiments.

Life history theory predicts that animals should increase their current reproductive effort as the probability of survival to the next reproductive opportunity decreases. We studied the effects of food depletion on life history and oviposition behavior in the egg parasitoid Paratelenomus saccharalis (Hymenoptera: Platygastridae) to determine if the parasitoid alters its reproductive strategy based on life expectancy. We first examined survivorship and reproductive effort of females provided only with water (i.e. starved) and females fed honey. Fed females lived up to 40 days, whereas starved females lived only up to 4 days. Fed females produced more offspring during life and had a higher net reproductive rate, i.e. R0, compared to starved females. Starved females did produce more offspring on the first day of emergence, however, and the intrinsic rate of natural increase, i.e. rm, did not differ between honey-fed and starved females. We further observed oviposition behavior in response to food availability during 24 h on the first day of emergence to determine the mechanism by which starved females increased reproductive output. Results showed that starved females oviposited more frequently than honey-fed females, and that the time required for a single oviposition was shorter for starved females. These results revealed that starved females had higher oviposition rates on the first day of emergence, leading to the similar rm of starved and fed females. This indicates that P. saccharalis altered their oviposition behavior to maximize their fitness under food-depleted conditions.

A new method for the simultaneous determination of two sweeteners (Advantame and Neotame) in processed foods using liquid chromatography (LC) with tandem mass spectrometry(MS/MS) was developed herein. Chromatographic separations were performed using an ACQUITY UPLC CSH C18 column at 40 °C via a mobile phase comprising 10-mmol/L ammonium formate and methanol. Samples were prepared via rapid dialysis using 30% methanol solution in a thermostatic shaker set at 160 rpm and 50 °C for 1 h. The matrix in the test solution had no effect on the identification and quantification of the compound without a clean-up step using solid-phase extraction (SPE). This method satisfied all validation criteria with a limit of quantification (LOQ) of 0.01 μg/g for all samples. Using this method, the amounts of Advantame and Neotame in 24 processed foods were subsequently investigated, with the results indicating their detection beyond the lower LOQ. Moreover, a multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) method was developed and described to further enhance product-identification ability.

Avian-origin influenza viruses like H5N1 and H7N9 often cause severe symptoms with high mortality in humans. Animal models are useful for clarification of the mechanisms of pathogenicity of these infections. In this study, to expand the potential utility of the Northern tree shrew (Tupaia belangeri) for influenza virus infection, we assessed the pathogenicity of H5N1 and H7N9 avian influenza viruses in tupaia. Infectious virus was detected continuously from nasal, oral, tracheal, and conjunctival swab samples in the animals infected with these viruses. H5N1 influenza virus infection of tupaia caused severe diffuse pneumonia with fever and weight loss. In contrast, H7N9 influenza virus infection caused focal pneumonia. The severity of pneumonia was correlated with proinflammatory cytokine transcript levels. These results indicated that tupaia can be another suitable animal model for avian influenza virus research.

Pupal parasitoids are considered to be idiobionts. However, some pupal parasitoids do not seem to kill or paralyze hosts at oviposition, including Tetrastichus brontispae, an endo-pupal parasitoid of Brontispa longissima. We conducted laboratory experiments to determine if T. brontispae parasitizes pupal hosts with a koinobiont developmental strategy. For this to be the case, its immatures would have to develop during the host transition from pupa to adult and in adulthood. With 0-d-old to 5-d-old pupal hosts, the T. brontispae females stung hosts of all ages with over 80% frequency. When the pupal hosts were parasitized, there were two modes of parasitism: pupal parasitism and pupal-adult parasitism. For pupal hosts that were parasitized, the majority showed a typical pupal parasitism, involving first the mummification of parasitized pupae and then the development of the parasitoid immatures within and emergence from these mummified pupae. However, the parasitized pupae that did not become mummified developed to host adults in which the parasitoid immatures developed, indicating pupal-adult parasitism. When parasitized pupal hosts were mummified, the 0-d-old to 3-d-old pupae were more likely than the older pupae to be mummified and had a greater number of parasitoid adults produced per host. The pupal-adult parasitism was not as successful. The parasitoid adults only successfully emerged from two host adults, which were parasitized as 2-d-old pupae, and one host adult parasitized as a 3-d-old pupa. The parasitoid eggs laid in 4-d-old and 5-d-old pupae continued to develop during and after the host transition from the pupa to adult. However, when the host adults died within 10 days after emergence, all the parasitoids died without completing their development. These results suggest that T. brontispae is a koinobiont parasitoid that successfully parasitizes young pupal hosts mainly through host mummification and rarely through pupal-adult parasitism. The fact that many parasitized 4-d-old or 5-d-old pupae died at the pupal or adult stage without producing any parasitoids, may also suggest that, under natural conditions, the pupal and adult mortality of B. longissima resulting from parasitism by T. brontispae is significant.

Intracellular bacteria that are mainly transmitted maternally affect their arthropod hosts' biology in various ways. One such effect is known as cytoplasmic incompatibility (CI), and three bacterial species are known to induce CI: Wolbachia, Cardinium hertigii, and a recently found alphaproteobacterial symbiont. To clarify the taxonomic status and provide the foundation for future studies to reveal CI mechanisms and other phenotypes, we investigated genetic and morphological properties of the third CI inducer that we have previously reported inducing CI in the coconut beetle Brontispa longissima. The draft genome of the bacteria was obtained from the oocytes of two isofemale lines of B. longissima infected with the bacteria: one from Japan (GL2) and the other from Vietnam (L5). Genome features of the symbionts (sGL2 and sL5) were highly similar, showing 1.3 Mb in size, 32.1% GC content, and 99.83% average nucleotide sequence. A phylogenetic study based on 43 universal and single-copy phylogenetic marker genes indicates that they formed a distinct clade in the family Anaplasmataceae. 16S rRNA gene sequences indicate that they are different from the closest known relatives, at least at the genus level. Therefore, we propose a new genus and species, "Candidatus Mesenet longicola", for the symbionts of B. longissima. Morphological analyses showed that Ca. M. longicola is an intracellular bacterium that is ellipsoidal to rod-shaped and 0.94 ± 0.26 μm (mean ± SD) in length, and accumulated in the anterior part of the oocyte. Candidates for the Ca. M. longicola genes responsible for CI induction are also described.

Habrobracon hebetor Say (Hymenoptera: Braconidae) is a cosmopolitan gregarious ectoparasitoid that attacks larvae of several species of Lepidoptera. Although there are two genetically different strains within H. hebetor, distribution of the strains has been poorly understood. In 2010, in Thailand, where H. hebetor has been known as a parasitoid of stored grain pests, it was found that H. hebetor attacked Opisina arenosella Walker (Lepidoptera: Oecophoridae), which is an invasive pest of coconut palm. For correct identification of this H. hebetor, we conducted DNA analysis and cross tests using populations collected from O. arenosella and stored grain pests in Thailand and populations in Japan known as H. hebetor. We obtained 413 bp of mitochondrial cytochrome oxidase I (COI) sequences and 414 bp of 16S rRNA gene sequences, and both indicated that there are two distinct clades within H. hebetor: one contains insects from Thailand, Spain, India, and Barbados; the other contains insects from Japan and the USA. There were no genetic differences or sexual isolation between Thai populations from different hosts. Our results also showed that populations in Thailand were sexually isolated from a H. hebetor population in Japan.

Maternally inherited bacterial endosymbionts in arthropods manipulate host reproduction to increase the fitness of infected females. Cytoplasmic incompatibility (CI) is one such manipulation, in which uninfected females produce few or no offspring when they mate with infected males. To date, two bacterial endosymbionts, Wolbachia and Cardinium, have been reported as CI inducers. Only Wolbachia induces complete CI, which causes 100% offspring mortality in incompatible crosses. Here we report a third CI inducer that belongs to a unique clade of Alphaproteobacteria detected within the coconut beetle, Brontispa longissima This beetle comprises two cryptic species, the Asian clade and the Pacific clade, which show incompatibility in hybrid crosses. Different bacterial endosymbionts, a unique clade of Alphaproteobacteria in the Pacific clade and Wolbachia in the Asian clade, induced bidirectional CI between hosts. The former induced complete CI (100% mortality), whereas the latter induced partial CI (70% mortality). Illumina MiSeq sequencing and denaturing gradient gel electrophoresis patterns showed that the predominant bacterium detected in the Pacific clade of B. longissima was this unique clade of Alphaproteobacteria alone, indicating that this endosymbiont was responsible for the complete CI. Sex distortion did not occur in any of the tested crosses. The 1,160 bp of 16S rRNA gene sequence obtained for this endosymbiont had only 89.3% identity with that of Wolbachia, indicating that it can be recognized as a distinct species. We discuss the potential use of this bacterium as a biological control agent.

Brontispa longissima (Coleoptera: Chrysomelidae) is a serious pest of coconut palm, and the species contains two cryptic species: the “Asian clade” is distributed over a wide area, including Asia and the Pacific region, whereas the “Pacific clade” is distributed in a limited area. Recent invasions and outbreaks have only been reported for the Asian clade, suggesting that invasive ability may differ between the clades. To reveal differences in invasiveness, we investigated the damage potential on coconut palm and range expansion of the two clades in Timor-Leste, where both clades are present. Distribution of the clades and of severely damaged trees indicated that range expansion and outbreaks have occurred for the Asian clade but not for the Pacific clade. The Asian clade attacked trees taller than 10 m, whereas the Pacific clade seldom attacked these trees. The preference for the taller trees, which are more abundant, can facilitate range expansion and outbreaks of the Asian clade. The beetle has spread through areas where coconut palms are continuously available, but have not expanded their distribution where coconut palms are separated by large gaps. This indicates that areas free of coconut palm could serve as buffer zones to prevent spread of this beetle.

Hepatitis B virus (HBV) infection causes acute and chronic hepatitis, which is a major public health concern worldwide. Immunization methods incorporating hepatitis B surface-small (HBs-S) antigen and hepatitis B core antigen (HBc) have been proposed as candidate therapeutic vaccines, but the elimination of existing HBV infection remains a challenge. To enhance the efficacy of HBs and HBc vaccination, we investigated HBs-large (HBs-L) as an immunogen, and carboxyl vinyl polymer (CVP) as an excipient. HBs-S or HBs-L, in combination with HBc antigen, was administered subcutaneously (without CVP) or intranasally (with or without CVP) for the evaluation of immune response in the tree shrew, which is considered to be a suitable small animal model of HBV infection. Immunization with HBs-L antigen by either route induced a rapid IgG response. Intranasal immunization with HBs-S or HBs-L and HBc formulated with CVP strongly induced neutralizing antibody activity, IgA response, and HBc-specific expression of the interferon gamma-encoding gene. These data indicated the potential of HBs-L and HBc intranasal immunization with CVP, not only as a therapeutic vaccine, but also as a prophylactic vaccine candidate.

The kudzu bug, Megacopta cribraria (Fabricius), is native to Asia but recently invaded the US and is expanding its distribution rapidly. To assess the probability of this bug traveling by attaching to the exteriors of fast-moving vehicles, we investigated the ability of M. cribraria adults to cling to stages with different surfaces (cloth, metal, or glass) against extreme airflows in a transparent acrylic tube connected to a vacuum cleaner. On the stages with cloth and metal, insects remained on the stages at 100 km/h wind speed. Estimated wind velocities required to blow 50 % of insects from the stages within 1 min were more than 100 km/h (cloth); 60 km/h (metal); and 40 km/h (glass) for males and 100 km/h (cloth); 50 km/h (metal); and 30 km/h (glass) for females. Together with frequent observations of attached M. cribraria on vehicle exteriors in the field, our results indicate a fairly high probability of range expansion of this species by attachment to vehicles.

In the present study, we demonstrate that the Japanese macaque (Macaca fuscata) can be used as an effective alternative in vivo model for investigating hypnozoite-induced relapsing infection caused by Plasmodium cynomolgi B strain, and that this model is comparable to the rhesus macaque model. Two female Japanese macaques (JM-1 and JM-2; aged 5 years; weighing about 4.0 kg) were used for the experiment. To produce sporozoites in mosquitoes, blood infected with P. cynomolgi B strain was collected from the donor monkey JM-1 and fed to approximately 200 mosquitoes using the standard artificial membrane feeding method. The isolated sporozoites (2 × 105) were intravenously inoculated into the JM-2 monkey, and the blood stage of the parasite was detected on day 8 after the infection. Chloroquine sulfate (CQ) was intramuscularly administered at a dosage of 6.0 mg/kg into the JM-2 monkey for 6 consecutive days from day 12 onward, after which the parasites disappeared from the peripheral blood. The first relapse occurred on day 26, which was treated again with CQ. Then, the second relapse occurred on day 44, which was cured by CQ treatment followed by the administration of primaquine phosphate (PQ) at a dosage of 1.0 mg/kg/day for 15 days. The JM-2 monkey was observed until 69 days after PQ administration, and there was no relapse during the entire follow-up period. We propose that the Japanese macaque model could contribute not only to drug screening for anti-hypnozoite activity, but could also be used as a powerful tool for investigating hypnozoite biology.

Copper oxides (CuO, Cu2O) have promising application potential in sensors or solar cells. In this study, copper oxide thin films were prepared by reactive DC magnetron sputtering using helicon plasma. A pure copper target was sputtered with Ar gas in an O2 atmosphere with a DC sputtering power in the range of 10–40 W, while the other fabrication conditions were kept constant. X-ray diffraction and x-ray photoelectron spectroscopy were used to investigate the effect of the sputtering power on the structure and the chemical state of the fabricated films. Atomic force microscopy was used to determine the relationship between the film's surface morphology and its structure. Hall effect measurements were employed to measure the semiconductor's properties, and the optical bandgap was determined by UV-Vis spectroscopy. The copper oxide film could be continuously tuned from n-type CuO to p-type Cu2O by changing the DC magnetron sputtering power.Copper oxides (CuO, Cu2O) have promising application potential in sensors or solar cells. In this study, copper oxide thin films were prepared by reactive DC magnetron sputtering using helicon plasma. A pure copper target was sputtered with Ar gas in an O2 atmosphere with a DC sputtering power in the range of 10–40 W, while the other fabrication conditions were kept constant. X-ray diffraction and x-ray photoelectron spectroscopy were used to investigate the effect of the sputtering power on the structure and the chemical state of the fabricated films. Atomic force microscopy was used to determine the relationship between the film's surface morphology and its structure. Hall effect measurements were employed to measure the semiconductor's properties, and the optical bandgap was determined by UV-Vis spectroscopy. The copper oxide film could be continuously tuned from n-type CuO to p-type Cu2O by changing the DC magnetron sputtering power.

A simple analytical method for the determination of hydrocortisone and progesterone in bovine, swine, and chicken muscle and eggs was developed. Hydrocortisone and progesterone were extracted with acetonitrile and subsequently cleaned-up using an Oasis HLB mini-cartridge. The method was validated in accordance with Japanese guidelines and exhibited trueness from 86.6% to 104.3% and precision (relative standard deviations (RSDs) of repeatability and within reproducibility were under 8.7% and 11.7%, respectively). The method was applied to 103 bovine muscle, 137 swine muscle, 69 chicken muscle and 52 egg samples that were commercially available in Tokyo, Japan. The hydrocortisone concentration was 0.9-41.2 µg kg(-1) in all bovine muscle samples, with an average of 7.7 µg kg(-1) and a median of 6.2 µg kg(-1). The progesterone concentration in 50 samples exceeded the limit of quantification (LOQ) and reached a maximum of 95.4 µg kg(-1). Hydrocortisone was also detected in all swine muscle samples at concentrations of 2.0-56.0 µg kg(-1). Its average and median concentrations amounted to 13.1 and 11.3 µg kg(-1), respectively. Twenty-three samples contained progesterone levels surpassing the LOQ, with a maximum concentration of 107.0 µg kg(-1). No chicken muscle samples contained any of the analytes. The progesterone concentration was 15.5-200.0 µg kg(-1) in all egg samples, with an average of 95.4 µg kg(-1) and a median of 90.5 µg kg(-1).

An accurate and selective analytical method for amantadine, which is used as antiviral drug to treat influenza A virus infection, was developed using LC-MS/MS. Residual amantadine was extracted from 4 kinds of food sample (poultry muscle, liver, gizzard and egg) with acetonitrile-pH 3.0 McIlvaine buffer (7 : 3), then cleaned up with an Oasis® MCX mini-cartridge. An external standard calibration curve was used for quantification, after sample purification by the combination of a reverse-phase strong cation exchange mixed mode cartridge for cleanup and a HILIC column for HPLC. The method was validated by performing recovery tests in accordance with Japanese guidelines for the validation of analytical methods for residual agricultural chemicals in food. Recovery ranged from 79.3% to 91.7%, RSDs of repeatability were under 3.3%, and RSDs of within-laboratory reproducibility were under 8.4%. This new method was applied to samples of poultry and egg purehased in Tokyo, but residual amantadine was not detected at all.

We developed a simultaneous determination method for 37 veterinary drugs in two chicken processed foods (deep-fried chicken and non-fried chicken cutlet) and muscle via liquid chromatography-mass spectrometry. The veterinary drugs belong to 7 different classes, including 4 antifolics, 4 benzimidazoles, 5 macrolides, 7 polyethers, 2 quinolones, 7 sulfonamides, and 8 other classes. The samples were extracted with ethyl acetate followed by acetonitrile with salt and buffers extraction. The two-step extraction enabled analyte extraction from highly lipid samples. The clean-up procedure, a solid-supported liquid extraction clean-up using a diatomaceous earth mini-cartridge, eliminated lipid co-extraction. The prepared sample matrix did not have an effect on the 36 analytes. The method was validated in accordance with the requirements of Japanese validation guidelines. Almost all targeted veterinary drugs successfully satisfied the guideline criteria in the three types of food matrices. The method exhibited recoveries of 70-105%, and the precision of repeatability and within-laboratory reproducibility ranged from 1 to 11% and 1 to 15%, respectively. The limits of quantification were estimated to range from 0.2 to 1.0μg/kg. Applying this method to samples commercially available in Tokyo, residues were detected in 3 out of 26 deep-fried chickens, 5 out of 20 non-fried chicken cutlets, and 17 out of 39 chicken muscles.

Bactrocera latifrons (Hendel) is believed to have originated in Southeast Asia but has invaded Hawaii and most recently East Africa. This insect has also been recorded on Okinawa Island, the far south of Kyushu Island, Japan. To assess the overwintering ability of B. latifrons adults, survival at constant temperatures (8, 10, 12, 14, 15 °C) and under fluctuating thermal regimes (FTRs) was investigated. At 14 or 15 °C, more than 30 % of females survived for 90 days. Time required to kill 95 % of B. latifrons at 8 °C was estimated to be 13 days; at 10 °C, 29 days; and at 12 °C, 38 days for females, and 8, 17, and 24 days at the same above temperatures, respectively, for males, suggesting low cold tolerance of this species. The results show that females survive cold temperatures better than males. Under an FTR of 11 °C (22 h)/20 °C (2 h) (average 11.8 °C) survival of females drastically increased compared to that at a constant temperature of 12 °C, whereas the survival of males increased slightly. Survival under FTRs indicates that adult B. latifrons may not overwinter in the north of Tanegashima Island, located 30 km south of Kyushu Island, Japan.

Rapid multi-residue analysis of pesticides in agricultural products was studied by using LC-MS/MS. Pesticide residues in 10 g of homogenized agricultural products were extracted with 30 mL of acetonitrile and salted out with 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride in the presence of citrate salts for buffering in a disposable tube. Co-extractives were removed by use of our original triple layered column (C18/GC/PSA; 60/30/60 mg). According to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan, we conducted recovery tests in 5 kinds of agricultural products (brown rice, kiwi, cabbage, sweet potato and spinach) spiked with 60 pesticides at the level of 0.01 or 0.1 μg/g. Each concentration of pesticide spiked was extracted from 2 samples per day on 5 days. Pesticides in the test solution were determined by two types of LC-MS/MS using scheduled MRM. Using this method, 58 out of 60 pesticides satisfied the guideline criteria in brown rice, 59 in kiwi, 55 in cabbage, 55 in sweet potato and 56 in spinach. This method is applicable for routine examination of pesticide residues in agricultural products.

To date, the chimpanzee has been used as the natural infection model for hepatitis B virus (HBV). However, as this model is very costly and difficult to use because of ethical and animal welfare issues, we aimed to establish the tupaia (Tupaia belangeri) as a new model for HBV infection and characterized its intrahepatic innate immune response upon HBV infection. First, we compared the propagation of HBV genotypes A2 and C in vivo in tupaia hepatocytes. At 8-10days post infection (dpi), the level of HBV-A2 propagation in the tupaia liver was found to be higher than that of HBV-C. Abnormal architecture of liver cell cords and mitotic figures were also observed at 8 dpi with HBV-A2. Moreover, we found that HBV-A2 established chronic infection in some tupaias. We then aimed to characterize the intrahepatic innate immune response in this model. First, we infected six tupaias with HBV-A2 (strains JP1 and JP4). At 28 dpi, intrahepatic HBV-DNA and serum hepatitis B surface antigens (HBsAg) were detected in all tupaias. The levels of interferon (IFN)-β were found to be significantly suppressed in the three tupaias infected with HBV A2_JP4, while no significant change was observed in the three infected with HBV A2_JP1. Expression of toll-like receptor (TLR) 1 was suppressed, while that of TLR3 and TLR9 were induced, in HBV A2_JP1-infected tupaias. Expression of TLR8 was induced in all tupaias. Next, we infected nine tupaias with HBV-A2 (JP1, JP2, and JP4), and characterized the infected animals after 31 weeks. Serum HBsAg levels were detected at 31 weeks post-infection (wpi) and IFN-β was found to be significantly suppressed in all tupaias. TLR3 was not induced, except in tupaia #93 and #96. Suppression of TLR9 was observed in all tupaias, except tupaia #93. Also, we investigated the expression levels of cyclic GMP-AMP synthase, which was found to be induced in all tupaias at 28 dpi and in four tupaias at 31 wpi. Additionally, we evaluated the expression levels of sodium-taurocholate cotransporting polypeptide, which was found to be suppressed during chronic HBV infection. Thus, the tupaia infection model of HBV clearly indicated the suppression of IFN-β at 31 wpi, which might have contributed to the establishment of chronic HBV infection.

A semi-transparent Cu thin film with a thickness of 40 nm and a tensile strength of 36 MPa onto the glass substrate deposited through a solution-based process, by heat-treating the spin-coated precursor film at 350 °C with Ar gas flowing at a rate of 1.5 L min−1. The Cu thin film is more than ca. 30% transparent in the visible region and exhibits 40−100% reflectance in the infrared region. The electrical resistivity of the Cu thin film is 4.7(6)×10−5 Ω cm.

A method of rapid analysis of multi-class residual veterinary drugs in milk, fish and shellfish was validated in accordance with Japanese guidelines for the validation of analytical methods for residual agricultural chemicals in food. Using LC-MS/MS, 43 multi-class veterinary drugs, including sulfonamides, quinolones, coccidiostats and antiparasites, could be analyzed in one injection. Analytes were extracted from samples with two kinds of solvent, acetonitrile containing 1 vol% formic acid and anhydrous acetonitrile, and salted out with 4.0 g of magnesium sulfate, 1.5 g of trisodium citrate and 2.0 g of sodium chloride. This method was assessed by performing recovery tests in retail milk and 4 kinds of fresh cultured fish and shellfish (salmon, tiger shrimp, red sea bream and bastard halibut) spiked with the 43 target analytes at the levels of 10 and 100 μg/kg. Using this method, 40 out of 43 drugs satisfied the guideline criteria in milk, 37 drugs in salmon, 42 drugs in tiger shrimp, 41 drugs in red sea bream and 39 drugs in bastard halibut.

The coconut hispine beetle Brontispa longissima (Gestro) supposedly originated in Indonesia and Papua New Guinea. It is a serious invasive pest of the coconut palm Cocos nucifera L. in Southeast and East Asia. In Japan, it has established itself using Satakentia liukiuensis (Hatushima) H.E. Moore as a main host on Ishigaki and Iriomote Islands where C. nucifera is rare. To assess the probability of further establishment of B. longissima in novel habitats where C. nucifera and S. liukiuensis are not available, we examined the suitability of four common palm species in Japan for oviposition and immature development of B. longissima: Chrysalidocarpus lutescens (Bory) H. Wendl., Phoenix roebelenii O'Brien, S. liukiuensis and Washingtonia filifera (Linden ex Andre) H. Wendl. When seedlings of the four palm species were placed in pots in an experimental field on Ishigaki Island, all four species were inhabited and infested by wild B. longissima adults. Oviposition and immature development were observed on P. roebelenii and S. liukiuensis but not on C. lutescens and W. filifera. When field-collected adults were released into mesh bags enclosing the potted seedlings, they oviposited on all four species. The eggs developed into adults on P. roebelenii, S. liukiuensis and W. filifera. On C. lutescens, however, hatched larvae died during the first or second instar.

An SRV-like virus was isolated from a colony-born Japanese monkey. To identify this SRV-like virus, we designed universal primers at regions that were conserved among the reported SRV sequences in the 5′-LTR and the short ORF and we obtained plasmid clones containing the complete gag, prt, pol and env genes. The full-length sequences of the isolate were determined from the plasmids and by direct sequencing. Sequence comparisons and phylogenetic analyses indicated that this SRV-like virus had a sequence identical to the reported 626 bp of SRV-5. In this study, we isolated SRV5/JPN/2005/V1 from a Japanese monkey and characterized the full-length SRV-5 sequence.

The larval parasitoid Asecodes hispinarum Boucek has been used to control the coconut hispine beetle, Brontispa longissima (Gestro) in Vietnam. Although A. hispinarum has succeeded in suppressing B. longissima in southern Vietnam, biocontrol has not succeeded in central Vietnam. One factor underlying this failure might be the high temperature during the hot season. In the present work, we examined whether A. hispinarum parasitises the beetles within the range of temperatures that occur during the hot season in central Vietnam and we evaluated the potential use of the pupal parasitoid Tetrastichus brontispae Ferriere. Development of the beetle and two parasitoids was investigated at two constant temperatures (28 and 30°C) and at fluctuating temperatures from 25 to 35°C (corresponding to temperatures during the hot season in central Vietnam). B. longissima reached adulthood in all the temperature treatments. More than 70% of A. hispinarum emerged from hosts at 28°C, but none emerged at 30°C or unde...

TiO2 thin films with a W layer to improve photo-functional properties were prepared using the reactive magnetron sputtering method. The W thin films at a thickness of 0.4 to 2.5 mm were coated onto the TiO2 thin films using the same magnetron sputtering. The surface chemical state of the W-coated TiO2 thin films measured by X-ray photoelectron spectroscopy showed W metal and/or W oxide on the TiO2 thin film. A spectrophotometer was used to investigate UV-Vis absorption of the W-coated TiO2 thin films which absorb energy of longer wavelength than the TiO2 thin film without the W surface layer. Visible-light photocatalysis of the W-coated TiO2 thin films was improved by four points in the sample with a W layer thickness of 1.7 nm, and photoconductivity was improved by four times under an artificial sun lamp. It is demonstrated that these improvements in the photo-functional properties were achieved by a different cause. © 2013 Wiley Periodicals, Inc. Electron Comm Jpn, 96(3): 45–52, 2013; Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ecj.11409

Brontispa longissima is a serious pest of the coconut palm Cocos nucifera, presumed to have originated in Papua New Guinea and Indonesia. It recently invaded Southeast and East Asia, where outbreaks have been reported. Mitochondrial DNA analysis reveals two cryptic species in B. longissima: one is distributed over a wide area including Asia and the Pacific region (the Asian clade) and the other in a limited area in the Pacific region (the Pacific clade). Recent invasions and outbreaks have been reported only from the area where the Asian clade has been found, suggesting that this clade has become a pest in Asia. To infer if the Asian clade has the ability to establish, spread and outbreak in novel habitats more effectively than the Pacific clade, we compared life-history traits between the two populations of different clades. The net reproduction rate (R0) was 130.0 and 94.0, the mean length of a generation (T) was 57.7 and 54.7 days, and the intrinsic rate of natural increase (r) was 0.084 and 0.083 per day for the population from Ishigaki Island, Japan (ISH) (the Asian clade) and for the population from Papua New Guinea (PNG) (the Pacific clade), respectively. Although the difference in r was little, the simulated population growth showed that the ISH population can be 1.6 times larger than that of the PNG after ten generations. The rapid population growth of the Asian clade would be partly responsible for its establishment, spread and frequent outbreaks in Asia.

Brontispa longissima (Coleoptera: Chrysomelidae) is a serious invasive pest of coconut palm (Cocos nucifera) supposedly originating from Indonesia and New Guinea. It has recently invaded Southeast and East Asia, where it has caused serious damage to coconut plants. Brontispa longissima as currently defined contains two cryptic species: we herein referred to one as the “Asian clade”, which is distributed over a wide area, including Asia and the Pacific region; and we referred to the other one as the “Pacific clade”, which is found in a limited area in the Pacific region. We developed a PCR–RFLP method for differentiating the two clades. Digestion of the PCR product of a 1,014-bp region within the mitochondrial cytochrome oxidase I gene (COI) with BslI, HpyCH4III, or NlaIV resulted in clade-specific patterns as estimated by the sequence data. We applied the method to specimens newly obtained from various locations to investigate the geographical distribution of B. longissima. Although B. longissima collected from Samoa in April 2003 had been placed in the Pacific clade, specimens collected from the same island in April 2010 were placed in the Asian clade, suggesting that the predominant clade may have been changing from the former to the latter. On Timor, specimens included both clades in apparently segregated habitats.

A transparent p-type Cu2O thin film of 50 nm thickness was successfully fabricated by means of a solution-based process involving the thermal reaction of molecular precursor films spin-coated on a Na-free glass substrate. The precursor solution was prepared by the reaction of an isolated Cu2+ complex of ethylenediamine-N, N, N′, N′-tetraacetic acid with dibutylamine in ethanol. The Cu2O thin films resulting from heat treatment of the precursor film at 450 °C for 10 min in Ar gas at a flow rate of 1.0 L min−1 were characterized by X-ray diffraction which indicated a precise cubic lattice cell parameter of a = 0.4265(2) nm, with a crystallite size of 8(2) nm. X-ray photoelectron spectroscopy peaks, attributable to the O 1s and Cu 2p3/2 level of the Cu2O film were found at 532.6 eV and 932.4 eV, respectively. An average grain size of the deposited Cu2O particles of ca. 200 nm was observed via field-emission scanning electron microscopy. The optical band edge evaluated from the absorption spectrum of the Cu2O transparent thin film was 2.3 eV, assuming a direct-transition semiconductor. Hall Effect measurements of the thin film indicated that the single-phase Cu2O thin film is a typical p-type semiconductor, with a hole concentration of 1.7 × 1016 cm−3 and hole mobility of 4.8 cm2 V−1 s−1 at ambient temperature. The activation energy from the valence band to the acceptor level determined from an Arrhenius plot was 0.34 eV. The adhesion strength of the thin film on the Na-free glass substrate was also determined as a critical load (Lc1) of 2.0 N by means of a scratch test. The method described is the first example of fabrication and characterization of a p-type Cu2O transparent thin film by a wet process.

A method for rapid analysis of multi-class residual veterinary drugs in livestock products was developed and validated in accordance with the Japanese guideline for pesticides. Using LC-MS/MS, 43 multi-class veterinary drugs, including sulfonamides, quinolones, coccidiostats and antiparasites, could be analyzed simultaneously in only 18 minutes. The extraction process was developed by modifying the QuEChERS approach to provide faster and less expensive extraction. The samples were extracted by using two kinds of solvent, acetonitrile and acetonitrile including 1 vol% formic acid, and salted out with magnesium sulfate, trisodium citrate and sodium chloride. Using these two extractants, 40 out of 43 drugs satisfied the guideline criteria in bovine muscle and swine muscle, 39 drugs were found in chicken muscle, and 37 drugs were found in eggs. The limit of quantification was less than the MRL for all analytes.

An analytical method for the determination of fluopicolide in livestock products and seafood was developed using LC-MS/MS. Sodium chloride was added to livestock products and seafood samples and fluopicolide was extracted twice with acetone after acidification with formic acid. The fat from the crude extract was removed using a macroporous diatomaceous earth column, followed by purification with a combination of mini-columns of GC (graphite carbon) and PSA (ethylenediamine-N-propyl silylation silica gel). The average recovery (n=5) of fluopicolide from 10 types of livestock products and seafood (cattle fat, cattle liver, cattle muscle, chicken, eel, egg, freshwater clam, honey, milk and salmon) spiked at the MRLs or at the uniform limit (0.01 ppm) was 96-100%, with a relative standard deviation of 2.3-6.2%. The limit of quantitation of the developed method was calculated to be 0.01 mg/kg.

To clarify the reproductive properties of the coconut palm pest, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae), we investigated in the laboratory the diel periodicity in copulation, effect of adult age after emergence on copulation and ovarian development, reproductive development in relation to female age, and the possibility of multiple mating. The beetles used in this study were reared in the laboratory and were fed an alternative food plant, narrowleaf cattail, Typha domingensis Pers. (Typhaceae). Copulation occurred throughout the 24-h cycle but mostly occurred in the photophase with two peaks, one at the onset of lights-on and the other 8–12 h after lights-on, indicating that most copulation activity in B. longissima in the field occurs in the early morning and the afternoon. Females could copulate immediately after emergence, whereas males began copulating 3 wk after emergence. Ovaries of females started to develop in the absence of mating and were correlated with adult age. Mature males could copulate with females regardless of ovarian developmental stage, and females with undeveloped ovaries accepted copulation attempts. Our study showed that the timing of copulation during the preoviposition period would not affect numbers of eggs laid, length of preoviposition period, or viability of eggs. B. longissima adults engaged in multiple mating.

The gregarious endoparasitoids Tetrastichus brontispae Ferriere is one of the important natural enemies of the coconut hispine beetle Brontispa longissima (Gestro), a serious invasive pest on coconut palm plants (Cocos nucifera L.) in Southeast Asia. Development at different temperatures, effect of host and female ages, effect of food and oviposition frequency and superparasitism were investigated in the laboratory. Females were allowed only one attack against one host in all experiments. The wasp developed in a host between 19 and 30°C, whilst no wasp completed its immature development at 16 and 31°C. Host and female ages affected parasitisation. Parasitoid emergence was high on day 0 and day 1 pupal hosts, and younger females produced more offspring than older females. The longevity of the female was affected not only by food supply, but also by oviposition frequency. The female survived longer when oviposition frequency was low. However, the total number of hosts parasitised by the female duri...

The coconut hispine beetle, Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae) is a serious pest of coconut palm. In this study, we developed an artificial diet for B. longissima so that the beetle could be used as a host for rearing two of its parasitoids, Asecodes hispinarum Boucek (Hymenoptera: Eulophidae) and Tetrastichus brontispae Ferrière (Hymenoptera: Eulophidae). The new artificial diet represents an improvement of our previous diet, which we used as a control. When beetle larvae were reared on the new diet, which contains cysteine but not cellulose powder and has twice as much coconut leaf powder as in the control, the adult emergence was 71% (approximately 2 times that in the control). We also examined the suitability of beetles fed on the new diet as hosts for the larval parasitoid A. hispinarum and the pupal parasitoid T. brontispae. The percentage of wasps that emerged from hosts that were fed the new diet was higher than that from the control-fed hosts. The new diet allowed both A. hispinarum and T. brontispae to produce adult wasps of the next generation, whereas the control only allowed T. brontispae to produce the next generation. These results suggest that the new diet is suitable for B. longissima and will facilitate mass-rearing of A. hispinarum and T. brontispae.

We developed a rapid and efficient means of determining residues of four nitroimidazoles—i.e., dimetridazole, ipronidazole, metronidazole, and ronidazole—and three hydrophilic metabolites—i.e., 2-hydroxymethyl-1-methyl-5-nitroimidazole, 1-methyl-2-(2′-hydroxyisopropyl)-5-nitroimidazole, and 1-(2-hydroxyethyl)-2-hydroxymethyl-nitroimidazole—in honey. We applied a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure improved to suit a nitroimidazole analysis, which is fast (approximately 30 min) and uses less organic solvent. The procedure involves initial single-phase extraction of 5 g of honey with acetonitrile containing 1% acetic acid, followed by liquid–liquid partitioning involving the addition of 5 g sodium chloride, 1.5 g trisodium citrate dihydrate, and 4 g magnesium sulfate. Moreover, matrix from honey was reduced by an SPE method with an alumina-N cartridge. The samples were analyzed using LC/MS/MS. Chromatographic separation of these nitroimidazoles and metabolites was performed in the gradient mode on a pentafluorophenylpropyl-bonded silica column (150 × 2.0 mm, 3 μm particle size) at 40°C. The mobile phase consisted of a 0.01% acetic acid solution and acetonitrile, and the flow rate was 0.2 mL/min. The method was validated using honey spiked with these nitroimidazoles from 0.1 to 0.5 μg/kg. The overall recovery of the seven nitroimidazoles ranged from 76.1 to 98.5%; intra- and interassay CV values were

A LC-MS/MS screening assay of multi-class antibiotics was developed for 19 residual antibiotics in livestock samples. Sample preparation employed the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach using 0.5% formic acid in acetonitrile-methanol (8 : 2), with salting-out using magnesium sulfate, trisodium citrate and sodium chloride. Recovery values from 5 different livestock samples ranged from 45.5 to 121.6%, and the RSDs were under 18% at two concentration levels. The limit of quantification values of 19 analytes were under 10 µg/kg in all livestock samples, and the procedure can detect almost all analytes under the MRL. Screening capability was confirmed by employing spiked samples. This new screening assay for residual antibiotics in livestock samples is expected to be useful for routine laboratory tests.

The coconut hispine beetle, Brontispa longissima (Gestro), supposedly originated from Papua New Guinea and Indonesia but has recently invaded Southeast and East Asian countries where it has been causing serious damage to Cocos nucifera L. This insect also occurs on the Southwest Islands off Kyushu Island in Japan. To evaluate the potential northward range expansion of this insect in Japan, we investigated its cold tolerance at 0, 5, and 10 °C (egg, larva, pupa, and adult), 13 °C (adult), and 15 °C (egg and hatched larva). At 15 °C, few eggs hatched, and the larvae that hatched died within a few days of hatching. At 13 °C, Ltime95 was estimated to be 23 days for adults, with the most cold-tolerant developmental stage at 10 °C. At all developmental stages, Ltime95 of B. longissima was estimated to be 19 days at 10 °C, 8 days at 5 °C, and 5 days at 0 °C, suggesting the cold tolerance of this beetle is very low. Considering average daily temperatures, it is unlikely that B. longissima can establish itself north of Amami-Oshima Island, located in the far south off the main island of Japan.

The coconut hispine beetle Brontispa longissima has been causing serious damage to the coconut palm Cocos nucifera in the Pacific, and Southeast and East Asia. This beetle also attacks Satakentia liukiuensis, an endemic palm on Ishigaki and Iriomote Islands, Japan. To assess the potential impact of B. longissima on S. liukiuensis, we examined the development period, survival rate, egg production and body size of B. longissima on S. liukiuensis at 22–31°C and consumption of S. liukiuensis leaf at 28°C. We also examined these life history traits on C. nucifera at 28°C to compare with those on S. liukiuensis. Brontispa longissima completed their development and reproduced on S. liukiuensis at 22–30°C. Linear regression analysis was used to estimate the effective cumulative temperature (k) as 891.2 degree-days, and the lower developmental threshold as 13.3°C. On the basis of these values and monthly average temperatures on Ishigaki Island, it was estimated that B. longissima has four generations per year on the island. Satakentia liukiuensis was less suitable for B. longissima than C. nucifera in terms of immature survival, development time, resultant adult size and reproduction. The low quality of S. liukiuensis as a host-plant may prevent the outbreak of B. longissima, which has not occurred yet on Ishigaki and Iriomote Islands.