DNA barcoding is a strong species identification tool for all animal taxa, and can easily be conducted when materials are under DNA friendly conditions. In contract, a full-length (659 bp) sequencing has been limited for the degraded DNAs extracted from old museum specimens. The initial challenges to retrieve the authentic DNA fragments from old museum specimens were attempted by obtaining short sequences (<300 bp) with the cloning process after PCR, making it both expensive and time-consuming. In this study, we employed a modified method to analyze the full-length DNA barcoding regions in 31~52 year-old butterfly specimens (301 dried specimens of 39 species) using direct sequencing after PCR with two different methods: 1) the successful PCR rates of 0 to 5.6% using four universal primer sets were too low to obtain authentic sequences and the cross-contamination was detected in almost all successful amplicons; 2) the success rates of PCR using specie-specific overlapping primer sets were distinctly high, reaching up to 75% with 98% authentic and 2% non-specific sequences. Thus, the result showed the method that using species-specific primer set per species yields the most effective success rates of both PCR and sequencing from degraded DNA without incorrect sequences.
We tested the identification ability of DNA barcodes comparing with morphological data using the Korean butterflies. The 921 samples (4.6 samples per species) for 202 resident Korean species except migratory species were used. The obtained samples were morphologically identified based on wing patterns. In a result, genetic divergence to the nearest-neighbouring taxon varied from 0 to 28.2%, with an average of 13.4 per cent. The neighbour joining (NJ) tree profile showed that sequence data for 185 of the 202 species formed distinct barcode clusters. Thus, our results indicated that 91.6 percent of the species were possible to allow the reliable identification using DNA barcoding. The rest 17 species (8.4%) consist of following four cases: clustering separated from each species by less than 1% branch length (two species pairs), paraphyletic clustering (two species pairs and one triple species pair), polyphyletic clustering with sharing barcodes (three species pairs), and clustering separated from existing species by the deep branch divergence (four clusters). However, it was not easy to interpret these ambiguous cases only using our current taxonomic evidences. Therefore, we are performing integrative taxonomy on these cases using other additional evidences such as examination on male genitalia and analysis of other gene regions.
Until now, seven species, Acosmeryx nega, Herpetogramma luctuosalis, Ostrinia furnacalis, Endoclyta excrescens, Spodoptera litura, Paranthrene regalis, and Nippoptilia vitis, have been known as lepidopteran grape pests in Korea (Woo, 1980; Lee, 1981; Kim, 1991; NIAST, 2002; Korea grape community, 2009). We discovered thirteen additional species belonging to eight families from Gwangwon and Chungbuk provinces, Korea. Additionally, we corrected the previous records of Deuterocopus albipunctatus and Nppoptilia vitis. They were identified by adult external or genitalic characters first, and also confirmed by the COI marker, compared with two public databases, NCBI and BOLD.
Relationship with fungi is one of the habitat adaptation of Sciaridae. While the earlier studies suggested that many fungus gnats are playing as the important vectors of various fungus diseases on plants and mushrooms, evidences have not been well reported. Based on a multi-gene phylogeny, by the Bayesian framework, we propose the correlation of four morphological characters of adults and habitat adaptation from saproxylic to phytophagous in the Sciaridae. Our results suggest that the evolution of habitat transition from dead plant litter to the live plant is related to habitat adaptation to the phytophagy and the morphological characters of fore tibia apex is also related to the functional linkage between these traits. We performed Bayes factor-based tests, referred with evolutionary pathway test (EPT), to decide the correlated traits gained the during evolutionary processes. The EPTs strongly suggest that fore tibia structures appeared first and followed by plant-feeding adaptation. The divergence time estimations of Sciaridae are also largely congruent with the fossil records. The members of subfamily Megalosphyinae have radiated explosively and contemporaneously since the Oligocene, with the expansion of modern grasslands and the increase of herbivores. Consequently, we suggest that the evolutionary benefit of tibial structure may be closely related with the fungal carrying in Sciaridae.
Metcalfa pruinosa (Say), native to North America, is spreading rapidly in the Korean Peninsula, causing serious damages on many deciduous forest trees, ornamental trees, and agricultural crops. Before the first report from Korea in 2005, M. pruinosa has not been reported from any other countries in the Eastern Palaearctic, while it has been record in Italy in 1979, and rapidly spreading into many European countries. To trace the invasion route of this species, we analyzed haplotype analysis of the mitochondrial cytochrome c oxidase subunit-I (mtCOI, 577bp), and developed microsatellite markers. In the haplotype analysis, a total of fourteen haplotypes were found from 69 individuals in 18 populations: 12 haplotypes (native region), 4 haplotypes (European region) and 2 haplotypes (Korea). Interestingly, Korean populations were clustered with some European populations. Eight polymorphic microsatellite loci were developed, and population structures were analyzed from 145 individuals in 8 populations. The origin and invasion route of M. pruinosa are under investigation.
The Diadegma fenestrale was known as parasitoid on potato tuber moth, Phthorimaea operculella and diamondback moth, Plutella xylostella. This species, genus Diadegma are first reported from Korea. DfIV showed typical ichnovirus shape which two membranes surround virus capsids. The genome contents of DfIV consist about sixteen double-stranded DNA segments ranging 2 to 6 kb. To identify DfIV genes, whole genome sequencing based on GS-FLX was conducted using purified total viral DNA extracted from D. fenestrale calyx. About sixty ORFs were analyzed and several typical polydna virus gene family detected such as cys-motif, rep, vinnexin and vankyrin. This is the first report of DfIV and these lepidopteran host immune suppression genes will be deeply identified.
We analyzed molecular and enzymatic properties of three cholinesterases (ChEs; ClAChE1, ClAChE2 and ClSChE) from Cimex lectularius. The ClAChE1 and ClAChE2 were generally present as a membrane-anchored dimeric insoluble form in the brain and ganglia. In the case of ClSChE, monomeric and dimeric soluble forms were observed. To investigate enzymatic properties, three ChEs were functionally expressed using baculovirus expression system. ClAChE1 revealed a significantly higher activity than ClAChE2 to acetylthiocholine iodide (ATChI) substrate. Kinetic analysis using two choline substrates (ATChI and butyrylthiocholine iodide) demonstrated that ClAChE2 had higher catalytic efficiency but lower substrate specificity than ClAChE1. Inhibition assay was conducted by using three inhibitors (BW284C51, eserine, Iso-OMPA) and two insecticides (chlorpyrifos-methyl and carbaryl). Two ClAChEs revealed high sensitivities to BW284C51, eserine, chlorpyrifos-methyl and carbaryl, but were not sensitive to Iso-OMPA. This inhibition profile confirmed that both ClAChEs are categorized as ChEs. Interestingly, the salivary specific cholinesterase did not show any measurable activities to choline substrates, confirming its non-synaptic function in C. lectularius
Parasitization by an endoparasitoid wasp, Cotesia plutellae, extends a larval period of Plutella xylostella and inhibits a larva-to-pupa metamorphosis. To determine antimetamorphic parasitic factor(s) in this host-parasitoid interaction, an effect of its symbiotic polydnavirus, Cotesia plutellae bracovirus (CpBV), was investigated by injecting purified virus particles to nonparasitized larvae of P. xylostella. Larvae injected with CpBV exhibited antimetamophosis in a viral dose-dependent manner. Also, the susceptibility to the viral injection was increased at young larval stages. Parasitized or virus-injected larvae shwed significant decrease in cell size of prothoracic gland and reduction in expression of ecdysone receptor (EcR) gene. However, they increased and maintained expression of insulin receptor (InR) gene. Twenty four CpBVsegments were individually injected to nonparasitized larvae. Only two segments (S22 and S27) had significant antimetamorphic effect. Subsequent RNA interference using double stranded RNA (dsRNA) was performed in each of encoded genes in each segment. Protein tyrosine phosphatase, ELP, and three hypothetical genes were determined to be antimetamorphic factors.
Sound treatments have been considered as a non-chemical insect pest control technique. Different frequency and intensity sounds were applied to immune and adult stages to screen any stress sounds to alter physiological processes. At 95 dB, 5,000 Hz and 30,000 Hz were selected to be stress sounds in audible and inaudible sound ranges, respectively. Both stress sounds significantly inhibited larval and pupal development. In biochemical analyses, lipid and sugar levels in plasma signigicantly increased in response to the stress sound treatments. Moreover, a digestive phospholipase A2 enzyme activity in midgut was significantly reduced. In adult stage, ultrasound treatment significantly inhibited mating behavior, which resulted in a reduced fecundity. These stress sounds altered gene expressions of stress-related genes, such as heat-shock proteins and apolipophorin III. This study suggests that extreme sounds play a role in physiological stress factors in S. exigua by altering developmental and reproductive processes.
The ant species, Vollenhovia emeryi, is distributed in Far East. The species can be divided into two major groups by their wing morphology of reproductives: short-winged and long-winged. A nationwide survey of the species was conducted for analyzing the mitochondrial haplotype diversity and genetic population structure. We collected 91 samples from 40 locations. A total of the 1239 bp partial COI (cytochrome C oxidase 1) region was used for the analyses. We found the total of 21 haplotypes. The mitochondrial haplotypes may correspond to the wing morphology. The genetic population structure examined potential geographic barriers of gene flow such as distance, mountains, rivers and plains which are non-mountain areas to prevent dispersal through mountain range. The result implied that no barriers considered in this study affected differently gene flow. Therefore, the behavioral characteristics of the ant may be the causal constraint of its genetic exchange.
Vollenhovia emeryi ant is distinguished by its wing morphology; short winged (SW) and long winged (LW). Its reproduction shows a bizarre genetic caste system distinct from other social hymenopteran insects. Unfertilized eggs undergo genome duplication and develop into clonal gynes. Fertilized eggs develop either into workers or males. The fate of the fertilized eggs is determined whether maternal genome loss (MGL) takes place after fertilization. Eggs with MGL become haploid males with only paternal half of the genome. Without MGL, the eggs become workers with maternal and paternal half of the genome. In this research, we analysed 5 nuclear genes of SW and LW individual ants. Among them, two genes from an SW male are identical to those of LW, and one gene from the SW male seems a variant of LW. The result indicates that SW males are derived from LW colonies. From the genetic relatedness point of view individuals in the same castes are genetically identical. On the other hand, between workers and two reproductives, the relatedness is asymmetrical and there is even no gene sharing between gynes and males. The conventional genetic relatedness by Hamilton is revised under this condition.
In haplodiploid sex determination, females are sexually reproduced from fertilized diploid eggs, and males from unfertilized haploid eggs. Haplodiploid sex determination seems simple in that sex depends simply on the ploid level. However, the underlying genetic mechanisms are thought to be much more complicated than expected. Among them, a powerful proposed mechanism is genomic imprinting. All epigenetic on-off systems require target genes, unless the systems target histone proteins on chromosomes. For Hymenoptera, a good candidate target gene in terms of sex determination is known either as feminizer (fem) or transformer (tra) in many insects. These two genes are essential for expressing femaleness. In most Hymenopteran insects, the maternal tra seems to be methylated and consequently not expressed, while the paternally derived tra gene is not methylated. Therefore, a fertilized egg with the paternally derived active tra gene will develop into a functional female. Like all Hymenoptera, ants (Formicidae) have haplodiploid sex determination. In Vollenhovia emeryi, however, queens are produced clonally while workers derive from fertilized eggs. Males are haploid, likewise deriving from fertilized eggs, but only after selective elimination of their maternal genome. Under the conventional genomic imprinting model, we would have expected that the opposite pattern of what is observed in others. Here we present extraordinary sex determination and suggest our hypothesis about genomic imprinting pattern in V. emeryi
A polydnavirus, Cotesia plutellae bracovirus (CpBV), is symbiotic to an endoparasitoid wasp, C.plutellae, which specifically parasitizes young larvae of the diamondback moth, Plutella xylostella. A recent study on CpBV replication by analysis of ovary transcriptome of C.Plutellae suggests several candidate coat protein genes. This study was conducted to confirm the coat protein genes by analyzing coat proteins of CpBV viral particles by a tandem mass MALDI-TOF. Immunoprecipitation of ovary protein extract with a polyclonal CpBV antibody captured three proteins named as p35, p60, and p70. More number of coat proteins were resolved in a protein extract directly from viral particles. All candidate coat proteins are analyzed in amino acid sequences by MALDI-TOF. A comprehensive analysis of viral proteomics and ovary transcriptome determined novel viral coat proteins from CpBV
An endoparasitoid wasp, Cotesia plutellae parasitized young larvae of diamondback moth, Plutella xylostella. Parasitized larvae exhibit sign ificant immunosuppression and fail to metamorphose to pupal stage. Especially, during last instar of parasitized P.xylostella, massive nutrients divert from host to wasp development. CpBV15α ,a host translation inhibitory factors encoded in C. Plutella bracovirus(CpBV), plays a crucial role in suppressing host usage of amino acids. Its promoter analysis shows that CpBV15α specifically inhibit host development in late larval period. To understand its inhibitory target, its specific expression was performed in non-parasitized P. xylostella by in vivo transient expression technique. Total plasma proteins were analyzed by 2D gel electrophoresis and determined target genes inhibited by CpBV15α. Immunoprecipation of cellular extract with CpBV15α antibody captured eIF2B. CpBV15α shares sequence homology with eIF5, especially at its eIF2B-binding region. Our results suggest that CpBV15α may sequester eIF2B, which result in malfunctioning of eIF2 cycling to form a translation initiation complex.
Teratocytes are originated from embryonic serosal membrane of some endoparasitoid wasps. Cotesia plutellae eggs release teratocytes in parasitoid host hemocoel at hatch in about 150 cells per egg. Teratocytes of C. plutellae were cultured in an insect culture medium for at least 14 days. Teratocytes cultured in vitro showed no increase in cell numbers but increased in cell size. Similarly,teratocytes in parasitized larvae did not increase cell numbers, but increased their cell size. Microinjection of invitro cultured teratocytes in to third instar larvae of nonparasitized Plutella xylostella showed a dose-dependently inhibitory effect on development and larval-pupal metamorphosis. In addition, teratocytes prolonged the immature developmental period and reduced the pupation rate, in which young aged host larvae were more sensitive to teratocytes treatment than old larvae. These results suggest that teratocytes play a crucial role in successful parasitization of C.plutellae by altering host developmental program.
As the immune reactions in human white blood cells of certain substances from insects to defend it when invaded by immune blood cells is increased. We experiment with changes in the total number of blood cells through the blood cells which increases and decreases, as well as to observe whether the immune response through any route is to evaluate what happens. Hemocyte population was analyzed in the last instar larvae of Spodoptera exigua. Granulocyte and plasmatocyte were predominant (>75%) types of hemocytes, whereas spherulocyte, prohemocyte, and oenocytoid hemocytes were observed in small densities (5~10%). Total hemocyte counts (THCs) were varied among different ages (day1-day5) of the last instar, in which day 3 larvae (L5D3) had the maximal density. Upon bacterial challenge to L5D3 larvae, THC was further enhanced within 2 h and then decreased to background level. This rapid THC increase in response to bacterial challenge was inhibited by injection with dexamethasone (1 ㎍ per larva). However, the addition of arachidonic acid reversed the inhibitory activity of dexamethasone and allowed the larvae to increase THC. This THC increase was mediated by cyclooxygenase products, but not by lipoxygenase products.
An entomopathogenic bacteria, Xenorhabdus nematophila (Xn) and Photorhabdus temperata subsp temperata (Ptt), suppresses insect immune responses and facilitates its symbiotic nematode development in target insect. Benzylideneacetone (BZA), PY, cPY, Ac-FGV, indole, 2-oxindole and 3-(4-hydroxyphenylpropionic) acid (PHPP) were compounds derived from the bacterial. Their immunosuppressive activities have been induced by inhibitory activity against eicosanoid biosynthesis and used to develop an additive to enhance control efficacy of other commercial microbial insecticides. This study investigated any cytotoxicity of their culture broth and bacterial metabolites on Spodoptera exigua hemocyte. When Xn or Ptt (<100 cells per larva) were injected to larval of S. exigua, the bacteria increased in density with incubation time, while the insent hemocyte numbers significantly and the resulting culture broths were sampled for analysis of their cytotoxicity against S. exigua hemocytes. In addition, the sequential culture broth samples were analyzed in active component chemicals using a reverse phase HPLC. Finally, seven bacterial metabolites were analyzed in relative cytotoxicity against S. exigua. These results suggest that BZA is a major cytotoxic compound.
Three tortricid pests, Grapholta dimorpha (Komai), G. molesta (Busck), and Carposina sasakii (Matsumura) are known as internal apple feeders in Korea. For identify young larvae which occurring serious damage in fruits, the molecular maker was developed from their mitochondrial DNA (mtDNA) sequences. To develop of PCR-RFLP marker, ND4 locus was digested with Swa Ⅰ. ND4-Swa Ⅰ digests showed two bands (396, 292 bp), one band (700 bp), and three bands (408, 178, and 103 bp) of G. dimorpha, G. molesta, and C. sasakii, respectively. Species-specific diagnostic PCR primers were developed in the ND4 locus and gave species-specific PCR products. Finally, these markers were applied to diagnose larvae infesting apples and showed species-specific fruit damage patterns, in which most feeders of G. dimorpha, G. molesta, and C. sasakii showed major feedings in apex, surface, and core of apple fruits, respectively.
The oriental fruit moth (Grapholita molesta) and the plum fruit moth (G. dimorpha) are internal feeders of apples. Their sympatric and similar sex pheromone compositions suggest their recent divergence in speciation. This study aims to determine genetic factors in this speciation by comparing transcriptomes associated in sex pheromone biosynthesis in these sibling species. Total RNAs were collected two female abodominal tips and read by a short read deep sequencing technology using an lllumina HiSeq. Almost 3-4 Gb reads were de novo assembled and resulted in 76,361 contigs of G. dimorpha and 104,463 contigs of G. molesta. More than 70% of these contigs were annotated and classified by a typical GO analysis. Transcriptomes related with sex pheromone biosynthesis were selected and grouped into fatty acid synthase, fatty acid oxidation. These analyses identified sex pheromone biosynthesis machineries
Male pheromone production and female reproduction of R.pedestris were evaluated on two different kinds of foods; sweet (non-astringent) persimmon and soybean. Male adults fed on soybean produced all the four pheromone components, (E)-2-hexenyl (Z)-3-hexenoate, (E)-2-hexenyl (E)-2-hexenoate, tetradecyl isobutyrate (C14iBu), octadecyl isobutyrate (C18iBu), whereas those fed on sweet persimmon did not produce C14iBu which is a key component in the function of the pheromone, and C18iBu. Female adults fed on soybean produced eggs, however, those fed on sweet persimmon did not at all. From these results, we concluded that host resource greatly affects the chemical communication and reproduction of both male and female of R.pedestris, and that sweet persimmon is not a proper food for its completion of life cycle.