Ting-Hsuan Hung

Molecular Identification of Forensically Important Blow Fly Species (Diptera: Calliphoridae) in Taiwan

Abstract Forensic entomology is a discipline that mainly uses insects collected in and around corpses to estimate the post-mortem interval in medicocriminal investigations. Among all scavenger and necrophagous insect groups that are related to corpses, blow flies (Diptera: Calliphoridae) are probably most important, not only because they occur in abundant numbers but also because they are one of the earliest groups to find corpses. However, most entomological evidence is strongly dependent on accurate species identification. Because identification allows the proper developmental data and distribution ranges to be applied in criminal investigations, species in Taiwan were surveyed from early 2000 and were identified using molecular data. Currently, eight species have been identified: Chrysomya megacephala (Fabricius), Chrysomya pinguis (Walker), Chrysomya rufifacies (Macquart), Hemipyrellia ligurriens (Wiedemann), Lucilia bazini Séguy, Lucilia cuprina (Wiedemann), Lucilia hainanensis Fan, and Lucilia prophyrina (Walker). We focused on classifying these blow fly species to establish a knowledge basis for further forensic entomological research in Taiwan. Because molecular data are helpful in identifying insect specimens, especially when no specimen of suitable condition for morphological identification is obtained, we extracted mitochondrial cytochrome oxidase subunit I (COI) DNA of the preceding blow fly species to study its application value for their differentiation. The cloning and sequencing of the COI gene (≈1,588 base pairs) of these eight species were completed, and the data were analyzed. Preliminary results revealed the high support of congeneric groupings of species by using COI data; these sequences were also shown to be highly conserved within the same species. To actually use the database of COI sequences under various specimen conditions, specific primers were also applied for different insect stages, different segments of adults, and specimens preserved for various times. A molecular primer key was ultimately constructed for the purpose of rapid and accurate species identification at the molecular level regardless of which stage or which part of a blow fly specimen is collected.

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis *

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.

Multiple Domains of the Tobacco mosaic virus p126 Protein Can Independently Suppress Local and Systemic RNA Silencing

Small RNA-mediated RNA silencing is a widespread antiviral mechanism in plants and other organisms. Many viruses encode suppressors of RNA silencing for counter-defense. The p126 protein encoded by Tobacco mosaic virus (TMV) has been reported to be a suppressor of RNA silencing but the mechanism of its function remains unclear. This protein is unique among the known plant viral silencing suppressors because of its large size and multiple domains. Here, we report that the methyltransferase, helicase, and nonconserved region II (NONII) of p126 each has silencing-suppressor function. The silencing-suppression activities of methyltransferase and helicase can be uncoupled from their enzyme activities. Specific amino acids in NONII previously shown to be crucial for viral accumulation and symptom development are also crucial for silencing suppression. These results suggest that some viral proteins have evolved to possess modular structural domains that can independently interfere with host silencing, and that this may be an effective mechanism of increasing the robustness of a virus.

Unusual Events Involved in Banana bunchy top virus Strain Evolution

Banana bunchy top virus (BBTV) can be transmitted by aphids and consists of at least six integral components (DNA-R, -U3, -S, -M, -C, and -N). Several additional replication-competent components (additional Reps) are associated with some BBTV isolates. A collected BBTV strain (TW3) that causes mild symptoms was selected to study the processes in BBTV evolution. Southern blot hybridization, polymerase chain reaction (PCR), and real-time PCR did not detect DNA-N in TW3. Real-time PCR quantification of BBTV components revealed that, except for the copy number of TW3 DNA-U3, each detected integral component of BBTV TW3 was at least two orders lower than that of the severe strains. No infection was observed in plants inoculated with aphids, which were first given acquisition access to the TW3-infected banana leaves. Recombination analysis revealed recombination between the integral component TW3 DNA-U3 and the additional Rep DNA-Y. All BBTV integral components contain a replication initiation region (stem-loop common region) that share high sequence identity. Sequence alignment revealed that TW3 DNA-R, -S, -M, and -C all have a stem-loop common region containing a characteristic 9-nucleotide deletion found only in all reported DNA-N. Our data suggest that the additional Rep DNAs can serve as sources of additional genetic diversity for integral BBTV components.

Identification of the Chinese Box Orange (Severinia Buxifolia) as an Alternative Host of the Bacterium Causing Citrus Huanglongbing

The Chinese box orange (Severinia buxifolia) was shown by graft-inoculation and psyllid-transmission tests to be an alternative host of the bacterium causing citrus Huanglongbing (HLB). A PCR-based assay for detection of the HLB bacterium (HLBB) was used to monitor HLBB. In graft-inoculation tests, the Chinese box orange (CBO) grafted with HLBB-infected scions of Luchen sweet orange (LSO) were positive for HLBB, 2–3 months after grafting. The back-grafting test demonstrated that HLBB-infected CBO scions could transmit HLBB back to LSO hosts via grafting. In psyllid-transmission tests, psyllids (insect vectors) transmitted HLBB to CBO plants, in which HLBB could be detected 3–4 months after inoculation. Acquisition-access tests of psyllids revealed that HLBB-free psyllids can acquire HLBB from diseased CBO hosts and can transmit HLBB back to the LSO plants. A field survey verified the presence of HLBB-infected CBO plants in the vicinity of citrus orchards. In this paper, CBO is shown to be a susceptible host plant in which HLBB can exist and replicate. It is also a donor plant from which HLBB can be transmitted to citrus hosts by grafting or by psyllid vectors.

Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and Citrus exocortis viroid infecting citrus in Taiwan

BackgroundTwo citrus viroids, Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), have been reported and become potential threats to the citrus industry in Taiwan. The distributions and infection rates of two viroids have not been investigated since the two diseases were presented decades ago. The genetic diversities and evolutionary relationships of two viroids also remain unclear in the mix citrus planted region.MethodsMultiplex RT-PCR was used to detect the two viroids for the first time in seven main cultivars of citrus. Multiplex real-time RT-PCR quantified the distributions of two viroids in four citrus tissues. Sequence alignment and phylogenetic analysis were performed using the ClustalW and MEGA6 (neighbor-joining with p-distance model), respectively.ResultsHSVd was found more prevalent than CEVd (32.2% vs. 30.4%). Both CEVd and HSVd were commonly found simultaneously in the different citrus cultivars (up to 55%). Results of the multiplex quantitative analysis suggested that uneven distributions of both viroids with twig bark as the most appropriate material for studies involving viroid sampling such as quarantine inspection.Sequence alignment against Taiwanese isolates, along with analysis of secondary structure, revealed the existence of 10 and 5 major mutation sites in CEVd and HSVd, respectively. The mutation sites in CEVd were located at both ends of terminal and variability domains, whereas those in HSVd were situated in left terminal and pathogenicity domains. A phylogenetic analysis incorporating worldwide viroid isolates indicated three and two clusters for the Taiwanese isolates of CEVd and HSVd, respectively.ConclusionsModerately high infection and co-infection rates of two viroids in certain citrus cultivars suggest that different citrus cultivars may play important roles in viroid infection and evolution. These data also demonstrate that two multiplex molecular detection methods developed in the present study provide powerful tools to understand the genetic diversities among viroid isolates and quantify viroids in citrus host. Our field survey can help clarify citrus-viroid relationships as well as develop proper prevention strategies.

First report of fruit rot disease of mango caused by Botryosphaeria dothidea and Neofusicoccum mangiferae in Taiwan.

Mango (Mangifera indica L.) is an economically important fruit crop in the tropical and subtropical areas of the world. In southern Taiwan, mango is grown on 18,000 ha of hilly land mainly located in Tainan, Kaohsiung, and Pingtung. Tons (180,000) of mango with a value of NT

First Report of a Fruit Rot Disease of Avocado Caused by Neofusicoccum mangiferae

6.6 billion (US

Cochin China atalantia (Atalantia citroides) as a new alternative host of the bacterium causing citrus Huanglongbing

206 million) are produced annually. In 2008, mango fruit rot disease was observed 1 week after harvest on 30 to 72% of stored mangoes collected from seven orchards in southern Taiwan. The initial symptom was a small, brown lesion and rot symptoms advanced progressively. Two predominant fungi were isolated from the margin of lesions on acidified potato dextrose agar (PDA with lactic acid, pH 3.8). Isolates of each fungal type were transferred to 2% water agar containing sterilized pine needles and exposed to near UV light to induce sporulation. For the first fungus, conidia obtained from pycnidia were ovate, one-celled, and hyaline, with an average length and width of 12.93 ± 0.93 × 6.98 ± 0.40 μm and an average length/width ratio of 1.85. To confirm the identity of the fungus, PCR amplification by universal primers, ITS1/ITS4, and DNA sequencing of the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) were conducted. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (Accession No. GQ421486). It showed a sequence identity of 100% with Neofusicoccum mangiferae (Syd. & P. Syd.) Crous, Slippers & A. J. L. Phillips) (GenBank Accession No. AY615185). For the second fungus, conidia obtained from pycnidia were fusiform, one-celled, and hyaline, with an average length and width of 22.87 ± 1.32 × 6.42 ± 0.46 μm and a length/width ratio of 3.53. The ITS sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (Accession No. GQ421485). It showed a sequence identity of 100% with Botryosphaeria dothidea (Moug.: Fr.) Ces & De Not.) (GenBank Accession No. AY 786321). To test pathogenicity, four mango fruits were wounded with a sterile needle, inoculated with mycelium agar plugs (0.5 mm in diameter) excised from separate monoconidial cultures, and incubated in a plastic box with a 100% relative humidity for 2 days at room temperature. Brown lesions appeared on all wounded sites of each fungus 2 days postinoculation. In control experiments, sterile agar plugs were placed on the wounded mango fruits. These fruits remained completely free from symptoms throughout the experiment. The pathogen was reisolated from the lesions of inoculated fruits and identified as N. mangiferae and B. dothidea, thus fulfilling Kochs postulates. N. mangiferae and B. dothidea have been reported on mango trees in Australia and South Africa (1). To our knowledge, this is the first report of these fungi causing fruit rot of mango in Taiwan. References: (1) B. Slippers et al. Mycologia 97:99, 2005.

Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts, rootstocks and the Asian citrus psyllid

Production of avocado (Persea americana) has increased significantly during the last 10 years in Taiwan and the area of cultivation is approximately 500 ha. The most important postharvest disease of avocado is anthracnose caused by Colletotrichum gloeosporioides (Penz.) in Taiwan (1). In 2008, a new disease was found to be infecting avocado fruit at some orchards in Tainan County of southern Taiwan. Infected avocados developed smooth, brown, circular spots first on the surface of harvested fruits. A fungus was always isolated from the margin of lesions and could also be found from symptomless fruit pedicles and stems. Fungal colonies cultured on acidified potato dextrose agar (PDA with lactic acid; pH 3.8) were initially colorless, turned dark gradually, and ultimately became gray to dark gray. After 4 days under fluorescent light at 25°C, pycnidia formed on PDA. Conidia obtained from fruiting bodies were ovate, one celled, and hyaline, with an average length and width of 12.9 (9.9 to 15.6) × 6.4 (5.2 to 7.2) μm. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (No. EU847427). It showed a sequence identity of 99% with Neofusicoccum mangiferae ((Syd. & P. Syd.) Crous, Slippers & A.J.L. Phillips) (GenBank No. AY615185). Thus, both morphological and molecular results confirmed the isolated fungus as N. mangiferae. Five avocado fruits were used to test the pathogenicity with three different treatment inoculation sites on each fruit. Wounded and unwounded sites on fruit were inoculated with mycelia agar plugs (0.5 mm in diameter) excised from a monoconidial culture and the fruit was kept in a plastic box with high humidity for 2 days at room temperature. Brown lesions appeared on all wounded sites 2 days postinoculation (dpi) and on unwounded sites at 4 dpi. The pathogen was reisolated from the lesions of inoculated fruits and found to be N. mangiferae, thus fulfilling Kochs postulates. In control experiments, sterile agar plugs were placed on the wounded avocado fruits. These fruits remained completely free from symptoms throughout the experiment. Several species of Botryosphaeria have been reported on avocado, including N. parvum (anamorph of B. parva), Fusicoccum aesculi (anamorph of B. dothidea), and Dothiorella aromatica (= F. luteum). To our knowledge, this is the first report of N. mangiferae causing fruit rot of avocado in Taiwan. Previously, N. mangiferae has been reported on mango trees worldwide, especially in Australia and Thailand (2). The presence of N. mangiferae in the subtropical area presents a serious disease problem not only to avocado but also to mango. References: (1) Y. P. Tsai, ed. List of Plant Diseases in Taiwan. 4th ed. Taiwan Phytopathological Society, 2002. (2) B. Slippers et al. Mycologia 97:99, 2005.