The bluetongue is a serious vector-borne viral disease that affect wild and domestic ruminants. It is transmitted by midges belonging to the genus of Culicoides which count more than 1350 species. Since 1998, the disease has been spread in Europe and North Africa in 2000 including Algeria, while Korea reported serotype 1 of the virus on 2015. To know further about the existence and distribution of Culicoides species in Algeria, adult midges were collected from 17 different regions in Algeria from 2009 to 2015. The Interactive Identification Key of Culicoides (IIKC) has been used, followed by molecular identification by the sequencing of partial COI gene. At least 21 genetically different species have been identified. Among them, at least 5 potential virus vector species are present in Algeria. This study provides important insights for the understanding of genetic diversity of Culicoides and the existing potential of bluetongue spread in Algeria.

Five heat shock protein 70 (hsp70) isoforms (hsc70-3, hsc70-4, hsc70-5, hsc70cb, hsp70Ab) of Apis mellifera were identified from Honeybee genome database. Specific primers for each isoform were designed for the quantitative realtime RT-PCR analysis, then analyzed transcript levels of the abdomen of adult workers (3-4 weeks old) in respond to heat shock and imidacloprid ingestion. Heat shock at 45°C for 1 h induced all 5 genes but highest in hsc70cb, hsp70Ab. Ingestion of imidacloprid pesticide did not change any hsp70 isoforms at 33°C but those bees also highly responsive to heat shock at 45°C. In addition, expression level of each hsp70 isoform was various and heat shock response of each isoform was tissue-specific. For example, hsc70-3 was highest in midgut, hsc70-4 was in hypopharyngeal gland, but hsp70Ab was in fat bodies. However, heat shock response of all 5 isoforms was the highest in the fat body than brain, hypopharyngeal gland, midgut, flight muscle and integument. Our results provide information for the understanding of complicated protective mechanism of honey bee against thermal stress.

The sweetpotato whitelfy Bemisia tabaci distribute worldwide and infests more than 500 species of plants. To determine nutritional stress of whiteflies at molecular level, we identified a full cDNA of glucose regulated protein 78 (grp78) which is known to be respond to nutritional restriction in vertebrate species. GRP78 of B. tabaci was highly conserved motifs of the HSP70 family and the C-terminal motif of KDEL characteristic of endoplasmic reticulum-specific HSPs. Real-time RT-PCR analysis showed that the grp78 level was not changed by thermal stress treatment from 4°C to 40°C for 1 h. However, the grp78 level was proportionally increased to the ingestion of a sucrose solution ranging in concentrations from 0% to 30% in a Parafilm feeding chamber. In addition, the grp78 levels were various by the ingestion of leaves of 10 different plants for 24 h; its level was higher with eggplant and pepper but lower with rice and apple. Our study suggests that the grp78 may have a role for cellular chaperones in relation to nutritional uptake of B. tabaci.

Indianmeal moth Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) is an economical pest of stored grains and their products, causing severe loss by feeding and producing silken web containing frass and exuvia. An ectoparasitoid Bracon hebetor Say (Hymenoptera, Braconidae) attacks this pest as a natural enemy and induce paralysis and developmental arrest in it. At molecular level its venom induce many physiological changes in host P. interpunctella to make it suitable source of food and development for its young ones. To explore these physiological changes at molecular level in P. interpunctella, we observed expression level of different genes having different functions related to immunity, defense and development at different intervals followed by B. hebetor envenomation. Fifth instar day 5 old larvae of P. interpunctella were used in experiment. Our results showed that B. hebetor envenomation effect the gene expression differently in host P. interpunctella. This basic study will be the starting point to understand the role of envenomation in host regulation studies.

Cotton aphid infests more than 700 plants and a major pest of various horticultural crops worldwide. The glucose regulated protein 78 (GRP78) is a member of heat shock protein 70. Its expression is associated with the nutritional changes as well as environmental stresses. The full sequences of grp78 cDNA of Aphis gossypii was determined. It had conserved motifs of hsp genes and terminated in KDEL which is common to GRP78. Quantitative realtime PCR showed that its level was changed during development and also upregulated by starvation. However, its level was not much changed by heat stress. The level of grp78 can be use to understand nutritional physiology on insects.

The sweetpotato whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a disastrous pest in horticultural plants worldwide. B. tabaci is a species complex including at least 24 biotypes in the world. In Korea, B-biotype has been invaded in 1998, Q-biotype in 2005 and widely spread into the country. B. tabaci is also a vector of more than 100 plant viruses, especially begomoviruses. Since 2008, Tomato yellow leaf curl virus (TYLCV) has been invaded into Korea and severely damaged tomato cultivar in all over of the country. B. tabaci is the only vector insect of TYLCV. Here we demonstrated whether TYLCV influence on the vector physiology during virus transmission. Pesticide susceptibility of whiteflies on TYLCV acquisition was determined in B. tabaci using the two-layered parafilm feeding chamber which containing 20% sugar solution including different doses of imidacloprid. Our result showed that TYLCV-viruliferous whiteflies were more susceptible to imidacloprid ingestion than non-viruliferous whiteflies. This study suggests that plant virus can manipulate the physiological conditions of vector insects.

The sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) is a species complex, including at least 24 biotypes which express different ecological and physiological characteristics. B. tabaci is one of the most serious pests in the horticultural crops in worldwide level. B. tabaci infests over 600 plant species and also indirectly damages plants by the honeydew excretion. Moreover, whiteflies transmit more than 100 plant viruses, especially begomoviruses. Tomato yellow leaf curl virus (TYLCV) which infested severely in tomato cultivars is transmitted by the vector insect, B. tabaci. Here, we demonstrated whether gene expression of B. tabaci is regulated by the oral ingestion of dsRNA. Double strand RNA (dsRNA) of heat shock protein 70 gene (hsp70) was produced from cDNA by using the specific primer. Whitefly adults were allowed to ingest sugar solution containing dsRNA for 3 hr in the two-layered parafilm feeding tube. Quantitative realtime PCR showed that whiteflies which ingest hsp70 dsRNA completely knockdown its transcript expression. Whiteflies ingestded dsRNA increased mortality by heat shock but not by cold shock. Further research will focus the role of hsp70 in various environmental stresses against insects.

Indianmeal moth Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) is an important pest of stored grains products. As a natural enemy, an ectoparasitoid Bracon hebetor Say (Hymenoptera, Braconidae) has been used to control Lepidopteran pest insects. Venom from parasitoid female alters many physiological functions in host insects. However, mechanism of physiological response of host insects against envenomation and parasitization is not clear. Here we observed the effect of B. hebetor envenomation on the gene expression (shsp, hsp70, grp78 and hsp90) in P. interpunctella at different time intervals of post envenomation. Fifth instar day 5 larvae of P. interpunctella were used in experiment to observe the effect of envenomation. Our results showed that parasitoid envenomation affected the gene expression differently in host insect. This study will provide comprehensive insights on physiological and biochemical mechanism in host-parasitoid relationships.

Root-lesion nematodes, Pratylenchus sp., are recognized as one of the most important plant parasitic nematodes. They infest various crop plants and cause severe root damages which lead to the yield loss of agricultural crops. There are 72 species recorded in the world, but 14 species in Korea. Soil samples were collected from chrysanthemum cultivars in various regions. Morphological and molecular diagnosis firstly recognized two species as Pratylenchus kumamotoensis and P. pseudocoffeae in Korea. Morphologically, P. kumamotoensis characterized by pharyngeal gland lobes overlapping with intestine dorsally. P. pseudocoffeae, which is similar with P. coffeae, but pharyngeal gland lobes are longer than P. coffeae. In molecular diagnosis, nucleotide sequences of D2-D3 region (700-800 bp) within 28S rDNA and ITS region including ITS1, 5.8S and ITS2 (700-1,000 bp) between 18S and 28S rDNA were determined from collected samples. NCBI database comparison of D2-D3 of P. kumamotoensis and P. pseudocoffeae were 99% similar with those of corresponding species, respectively. ITS of P. pseudocoffeae was 99% similar within species. ITS sequence of P. kumamotoensis was firstly determined in this study. Distribution of P. kumamotoensis was only reported in Japan but P. pseudocoffeae is present in Japan and USA. Here we firstly report the presence of these two species in Korea.

Heating for disinfestation has been practiced of at the condition of 55-60°C for at least 48 hours in various facilities of stored and processed agricultural products. However, it has been required to reduce temperature and time due to the economic efficiency. To improve disinfestation efficiency of heating, we demonstrated whether combined treatments of heat with other agents exert synergistic for disinfestation. Heat treatments were combined with 1 ppm diatomaceous earth (DE), 10 ppm phosphine or 10% carbon dioxide against Plodia interpunctella, Sitophilus zeamais and Tribolium castaneum. Insects were treated single or multiple combinations of each component for 6 h at either 25°C or 40°C, then mortalities were monitored for 14 days post-treatments. Combined treatments greatly enhanced mortalities and shorten killing time of three species rather than single treatments. Particularly, heat treatments with 1 ppm DE and 10 ppm phosphine completely lethal within 3-6 h post-treatments, but heating with 10% CO2 was not much effective. Among three species, S. zeamais adults were more suscpetable to heat but both larvae and adults of T. castaneum was highly susceptible to phosphine. Our results clearly showed that combined treatment of DE or phosphine with mild heating (40°C) greatly improved mortalities of three major stored pest insects and provide advanced techniques for disinfestation of stored product pests.

We provide the first report on Stigmaeopsis miscanthi Saito, which was identified from Chinese silver grass Miscanthus sinensis on Ulleung Island in Korea. This species is one of the S. celarius complex, which involves several cryptic species. S. miscanthi has longer second dorsal propodosomal setae (P2), which is an important characteristic for species identification in the genus Stigmaeopsis. We determined nucleotide sequences of the internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit I (COI) of S. miscanthi. The COI sequence differed by 0.6% between Korean and Japanese strains. The comparison between S. miscanthi and S. celarius showed that ITS2 and COI differed by 7.2% and 7.9%, respectively. In addition, species-specific primer sets of both species were designed to show the species classification within the genus.

Bemisia tabaci is a serious pest in various horticultural crops in the world. Due to use of chemical pesticide for their management they develop pesticide resistance and environmental contamination. It is necessary to develop alternative bio-pesticides using natural products from plants and natural enemies. Nicotiana benthamiana is a variety of wild tobacco plants and produce acyl sugars from glandular trichomes in the leaves. When adult whiteflies were reared with fresh N. benthamiana leaves, they were completely dead within 84 h. Oral feeding of 20% N. benthamiana extracts using ethanol and water showed complete mortality of whiteflies within 48 hours. Spray of N. benthamiana extracts into the leaves was lethal to eggs but not to nymphs of whiteflies. Further, tomato plants sprayed with N. benthamiana extracts were highly repellent to adult whiteflies. Quantitative real-time PCR indicated that the expression of various genes of B. tabaci was changed by oral feeding of N. benthamiana extract. This study suggests N. benthamiana extract is a useful for the control of whiteflies and can be used as an alternative natural pesticide for the whitefly management.

Acquisition of plant viruses has various effects on physiological mechanisms in vector insects. Bemisia tabaci is the only known vector of Tomato yellow leaf curl virus (TYLCV), which is a serious virus affecting tomato cultivars. In this study, the lifespan of Q1 biotype was compared between non-viruliferous (NV) and TYLCV-viruliferous (V) whiteflies. Total lifespan from egg to adult death of NV whiteflies was 62.54 days but 10.64 days shorter in V whiteflies. We investigated the temperature susceptibility of B. tabaciby comparing mortalities as well as heat shock protein (hsp) mRNA levels between NV and V whiteflies. For this, NV and V whiteflies were exposed for either 1 or 3 h at 4, 25, and 35 °C. The mortality of V whiteflies was higher than NV ones following exposure at either 4 or 35 °C, but there was no significant difference at 25 °C. Analysis of the expression level of heat shock protein (hsp) genes using quantitative real-time PCR showed that both cold and heat shock treatments stimulated higher expression of hsps (hsp40, hsp70, and hsp90) at various rates in V whiteflies than NV ones, but there was no difference at 25 °C. All together, our results show that TYLCV acquisition accelerated the developmental rate and increased susceptibility to thermal stress in B. tabaci. Therefore, this modification may result in reduced vector longevity due to increased metabolic energy utilization. Our results provide insights into the complex interaction between vector fitness and thermal stress in relation to the acquisition and transmission of plant viruses.

Bemisia tabaci is a serious pest in various horticultural crops in the world. The management of B. tabaci has been typically carried out by chemical pesticides. Due to the development of pesticide resistance and environmental contamination, however, it is necessary to develop alternative biopesticides using natural products from plants and natural enemies. Nicotiana benthamiana is a variety of wild tobacco plants and produce acyl sugars from glandular trichomes in the leaves. Acyl sugars are known to be highly toxic to various plant sapping insects such as whiteflies, aphids and thrips. Here, we extracted acyl sugars in two different ways from the leaves. At first, collected leaves were simply washed with water. Otherwise, collected leaves firstly dried and homogenized into the powder, then extracted with ethanol. Spray of 10% water-extracted solution into adult whiteflies showed 80% mortality. Otherwise, spray of 10% ethanol-extracted solutions showed complete mortality at 48 h after treatment and also strong repellency of adult whiteflies into the treated tomato plants. Our results suggest N. benthamiana is a useful for the control of whiteflies and can be used as an alternative natural pesticide for the whitefly management.

Plodia interpunctella, Indianmeal moth, is world-widely distributed pest in stored and manufactured agricultural products. Practically, this species seriously damaged by the infestation of most stored and manufactured agricultural products. It is necessary to find useful techniques for the control of stored-product pests insects including P. interpunctella. Recently, heating at 55℃ for 48 h can be practically used to disinfest stored-product insects in the filed of manufactures. To improve efficiency of this technique we conducted combination treatments with heat and diatomaceous earth. At room temperature, wandering larvae, which is most tolerant developmental stage to heat, were not dead regardless of diatomaceous earth (4 mg/L) treatment within 24 h. However, incubation at 40℃, all the larvae were dead with diatomaceous earth treatment while its mortality was 28% without diatomaceous earth treatment. Thus, heating effects was significantly improved by the combined treatments of diatomaceous earth. This effect was increased when temperature was higher and the exposed time is longer. Our results clearly showed that the combined treatments of both heat and diatomaceous earth showed synergistic effects for the control of P. interpunctella.

The genus Panonychus has been reported only two species, P. ulmi and P. citri, in Korea. Two new species, P. mori Yokoyama, 1929 and P. caglei Mellot, 1968 were firstly identified from jujube orchards in Gyeongsan and kudzu vine in Byunsan peninsula in Korea. Morphological differences among four species have been described especially in aedeagus shape. Comparison of nucleotide sequences of both the internal transcribed spacer 2 (ITS2) region of nuclear ribosomal RNA gene and the mitochondrial cytochrome oxidase subunit I (COI) gene were compared between four species. Phylogenetic analysis of ITS2 and COI sequences using neighbor-joining method showed that P. mori and P. caglei were most similar to each other and more closely related to P. ulmi than P. citri. In addition, species-specific primer sets of each species were designed based on ITS2 sequencesand can be used to diagnose species in this genus.

Field surveys for the B. tabaci species complex were conducted from 2009 to 2013 in Korea, and the results were compared with published data of the B. tabaci complex. Three species, Mediterranean, Middle East-Asia Minor 1, and JpL, were determined from several regions based on mitochondrial COI sequences. The Mediterranean was mainly collected in greenhouses, displacing the earlier invasive species, Middle East-Asia Minor, and the JpL was collected in the field. The JpL is newly confirmed as a unique species of B. tabaci complex in Korea and Japan.

Chrysanthemum stunt viroid (CSVd) is a serious pathogen affecting chrysanthemum that has caused significant economic losses to Chrysanthemum flower production worldwide. Control of CSVd disease is difficult due to its contagious nature and long latent period in the field. As chrysanthemum is most often produced by implanting seedlings, it is necessary to diagnose CSVd infection before cultivation. In this study, we screened CSVd infection in seedlings from 30 varieties including 5 domestic, 6 Japanese, and 19 European varieties. Molecular diagnosis of the combination of RT-PCR and nested PCR showed that CSVd was not detected by the first RT-PCR but detected by the second nested PCR analysis in 10 varieties, including 1 domestic, 2 Japanese, and 7 European varieties. Further comparison of 10 identified CSVd nucleotide sequences showed that those are highly conserved (99-100%) and the most similar to an isolate (AB006737) identified in Hokkaido, Japan. Our study suggests that the combination of RT-PCR and nested PCR analysis is successful for the CSVd diagnosis of seedlings and the molecular diagnosis is necessary to prevent the introduction and propagation of viroid disease into the fields.

The sweet potato whitefly, Bemisia tabaci (Gennadius, 1889) (Hemiptera: Aleyrodidae), is one of the most important agricultural pests by direct feeding and begomovirus transmission in the world. B. tabaci species complex consisting of about 30 species worldwidely has been mainly distinguished by molecular methods because of difficulties detecting morphological differences. In Korea, distribution of two exotic groups, the Mediterranean (Q biotype) and the Middle East-Asia Minor 1 (B biotype), were confirmed by comprehensive molecular methods in 2012. Of them, the Q biotype as a vector of Tomato yellow leaf curl virus (TYLCV) has caused significant crop yield losses. Tobacco leaf curl virus (TbLCV), one of four begomoviruses in Korea, was first reported in 2011. It damaged, although not frequent, to greenhouse tomatoes at Iksan, Gimje, Sunchang, and Jeju. The vector transmitting TbLCV, however, is not known yet. In this study, we sampled an unknown B. tabaci populations on leaves of the Japanese honeysuckle (Lonicera japonica) at Iksan in outdoor conditions. PCR analysis revealed that some populations were infected with TbLCV. We identified an unknown indigenous genetic group named as JpL and clarified its phylogenetic relationships based on nucleotide sequences of mitochondrial cytochrome oxidase I gene.

Vector-borne plant virus transmission is a complex mechanism involving various interactions of viruses, vectors and plants. Plant viruses modify, either directly or indirectly, the rates of development, reproduction and behavior of vector insects in a positive, negative, or neutral manner. The sweetpotato whitefly, Bemisia tabaci, is the only known vector of Tomato Yellow Leaf Curl Virus (TYLCV) which is a virus that seriously damaged tomato cultivars all around the world. Acquisition of TYLCV influences on various developmental, physiological and molecular mechanisms of Q1 biotype B. tabaci. We compared several behavioral and physiological characteristics between non-viruliferous (NV) and TYLCV-viruliferous (V) of Q1 biotype whiteflies. When B. tabaci acquired TYLCV, total life span was shorter and fecundity was lower than NV ones. V whiteflies were more susceptible to thermal stress by increasing hsp mRNA levels but higher in mortality by either heat or cold shocks. Furthermore, V whiteflies increased the rates of plant sap probing and light attraction behaviors than NV ones. All together, our results show that TYLCV acquisition regulates B. tabaci to enhance the rates of development and environmental susceptibility. Our results provide insights to understand vector’s role in relation to the acquisition and transmission of plant viruses.