Renius cornutus, a new genus and species of Chilocorini from Tibet, China (Coleoptera, Coccinellidae).

A new monotypic genus of Chilocorini, Renius Li & Wang, gen. n., with a new species R. cornutus Li et Wang, sp. n. is described from Tibet, China. All diagnostic features are illustrated. The relationships with other genera of Chilocorini are discussed and a key to Chinese genera of Chilocorini is provided.

A new species of the genus Phaenochilus Weise from China (Coleoptera, Coccinellidae, Chilocorini).

A new species Phaenochilus albomarginalis Li & Wang, sp. n. is described. The only other species recorded from China is Phaenochilus metasternalis Miyatake, 1970 and it is described here for comparison. Diagnoses, detailed descriptions, illustrations, and distributions are provided.

Discovery of a New World ladybird beetle Nephaspis indus Gordon, 1996 (Coleoptera: Coccinellidae: Scymnini) on the Island of Taiwan.

BACKGROUND: Nephaspis indus Gordon, 1996 was imported into Taiwan from Hawaii in 1990 as a biological control agent for the spiralling whitefly, Aleurodicus dispersus Russell, 1965 (Hemiptera: Aleyrodidae). However, its establishment was not known prior to this study. NEW INFORMATION: Nephaspis indus Gordon, 1996, a natural enemy of Aleurodicus dispersus Russell (Hemiptera: Aleyrodidae) native to the Neotropical region, is recorded as established in Taiwan for the first time. The present paper provides a detailed further description and illustrations of the adult. Diagnostic characters for the genus and species are given and the nomenclature of this species is also discussed.

A taxonomic review of the genus Horniolus Weise from China, with description of a new species (Coleoptera, Coccinellidae).

Five species of the genus Horniolus Weise, 1901 from China are revised, including the description of a new species, Horniolus hainanensis Chen & Ren, sp. n.Horniolus sonduongensis Hoàng, 1979 is reported from China for the first time. A key to the species from China is provided. Nomenclatural history, diagnoses, detailed descriptions, illustrations, and distribution for each species have been provided. A checklist of all known species of this genus is also presented.

Wolbachia Has Two Different Localization Patterns in Whitefly Bemisia tabaci AsiaII7 Species.

The whitefly Bemisia tabaci is a cosmopolitan insect species complex that harbors the obligate primary symbiont Portiera aleyrodidarum and several facultative secondary symbionts including Wolbachia, which have diverse influences on the host biology. Here, for the first time, we revealed two different localization patterns of Wolbachia present in the immature and adult stages of B. tabaci AsiaII7 cryptic species. In the confined pattern, Wolbachia was restricted to the bacteriocytes, while in the scattered pattern Wolbachia localized in the bacteriocytes, haemolymph and other organs simultaneously. Our results further indicated that, the proportion of B. tabaci AsiaII7 individuals with scattered Wolbachia were significantly lower than that of confined Wolbachia, and the distribution patterns of Wolbachia were not associated with the developmental stage or sex of whitefly host. This study will provide a new insight into the various transmission routes of Wolbachia in different whitefly species.

Revision of the subgenus Orthoscymnus Canepari of Scymnus Kugelann (Coleoptera, Coccinellidae), with descriptions of four new species.

The subgenus Orthoscymnus Canepari, 1997 of Scymnus Kugelann, 1794 is herein revised. Seven species of the Orthoscymnus fauna are recognized, of which four species, Scymnus (Orthoscymnus) jilongicus sp. n., Scymnus (Orthoscymnus) paradoxus sp. n., Scymnus (Orthoscymnus) crispatus sp. n. and Scymnus (Orthoscymnus) duomaculatus sp. n., are described as new to science. Scymnus (Orthoscymnus) rhododendri Canepari is recorded from China for the first time. Scymnus (Pullus) robustibasalis Yu is transferred to the subgenus Orthoscymnus (comb. n.). All species are diagnosed, described and illustrated, and distributions are provided for each species. A key to the species is included.

The Ifchit1 chitinase gene acts as a critical virulence factor in the insect pathogenic fungus Isaria fumosorosea.

The filamentous fungus, Isaria fumosorosea, is a promising insect biological control agent. Chitinases have been implicated in targeting insect cuticle structures, with biotechnological potential in insect and fungal control. The I. fumosorosea chitinase gene, Ifchit1, was isolated and determined to encode a polypeptide of 423 amino acids (46 kDa, pI = 6.53), present as a single copy in the I. fumosorosea genome. A split marker transformation system was developed and used to construct an Ifchit1 gene knockout. The ΔIfchit1 strain displayed minor alterations in mycelial growth on diverse media at 26 °C compared to the wild type and complemented (ΔIfchit1::Ifchit1) strains; however, colony morphology was affected, and the mutant strain had a temperature sensitive phenotype (32 °C). Although sporulation was delayed for the mutant, overall conidial production was almost twice than that of wild type. Biochemical assays indicated decreased chitinase activity during growth in Czapek-Dox liquid media for the ΔIfchit1 strain. Insect bioassays using diamondback moth, Plutella xylostella, larvae revealed decreased infectivity, i.e., increased LC 50 (threefold to fourfold) and a significantly delayed time to death, LT 50 from 3 to 6 days, for the ΔIfchit1 strain compared to the wild type and complemented strains. These data indicate an important role for the Ifchit1 chitinase as a virulence factor in I. fumosorosea.

Tumidusternus, a new genus of Aspidimerini from China (Coleoptera, Coccinellidae).

Tumidusternus gen. n., along with Tumidusternusfujianensis sp. n. (Coleoptera, Coccinellidae, Aspidimerini) from China is described and illustrated. A key to the tribe Aspidimerini is given.

Contribution to the genus Xanthocorus Miyatake (Coleoptera, Coccinellidae, Chilocorini).

The genus Xanthocorus Miyatake, 1970 consists of three species from China, including two new species described here: Xanthocorusnigrosuturalis sp. n. and Xanthocorusmucronatus sp. n. A key to identification of species is given. Diagnoses, detailed descriptions, illustrations, and distributions are provided.

Status of insecticide resistance and selection for imidacloprid resistance in the ladybird beetle Propylaea japonica (Thunberg).

Field populations or strains of Propylaea japonica collected from four places in southern China (Guangzhou, Nanning, Guilin, and Yuxi) were tested for susceptibility to four insecticides (abamectin, imidacloprid, beta-cypermethrin, and chlorpyrifos) by the Petri-dish Potter tower method and compared with an insecticide-susceptible strain. Concentrations that proved lethal for 50% of the tested individuals (LC50) were estimated by probit analysis, and resistance factors (RF) were calculated at the LC50 level, which ranged from 1.6 to 10.1, depending on the insecticide. In addition, the Guangzhou strain formed the original population for imidacloprid resistance selection. After selection for 20 generations, the resistance had increased 39.3-fold. Fitness analysis in terms of such traits as fecundity, days to maturity, and survival showed that although both resistant and susceptible populations developed at comparable rates, the resistant strain was less fecund (it laid fewer eggs and a smaller proportion of those eggs hatched and resulted in adults), attaining a fitness score of only 0.56 relative to the susceptible strain. These observations suggest that it is possible to detect strains of P. japonica highly resistant to insecticides under laboratory conditions, and that resistance to imidacloprid carries considerable fitness costs to P. japonica. The study served to expand our understanding of the impact of imidacloprid resistance on biological parameters of P. japonica in more detail and to facilitate the deployment of natural enemies resistant to insecticides in integrated pest management.

A contribution to Asian Afidentula Kapur (Coleoptera, Coccinellidae, Epilachnini).

Two new species of Afidentula, Afidentuladentata sp. n. and Afidentulajinpingensis sp. n. are described from China. Afissasiamensis Dieke is moved to Afidentula comb. n.. All three species are described and illustrated, and a distribution map is given. A key to Asian species of Afidentula is updated. Diagnostic similarities and differences between Afidentula and Afidenta are discussed and illustrated.

The intracellular bacterium Wolbachia uses parasitoid wasps as phoretic vectors for efficient horizontal transmission.

Facultative bacterial endosymbionts are associated with many arthropods and are primarily transmitted vertically from mother to offspring. However, phylogenetic affiliations suggest that horizontal transmission must also occur. Such horizontal transfer can have important biological and agricultural consequences when endosymbionts increase host fitness. So far horizontal transmission is considered rare and has been difficult to document. Here, we use fluorescence in situ hybridization (FISH) and multi locus sequence typing (MLST) to reveal a potentially common pathway of horizontal transmission of endosymbionts via parasitoids of insects. We illustrate that the mouthparts and ovipositors of an aphelinid parasitoid become contaminated with Wolbachia when this wasp feeds on or probes Wolbachia-infected Bemisia tabaci AsiaII7, and non-lethal probing of uninfected B. tabaci AsiaII7 nymphs by parasitoids carrying Wolbachia resulted in newly and stably infected B. tabaci matrilines. After they were exposed to infected whitefly, the parasitoids were able to transmit Wolbachia efficiently for the following 48 h. Whitefly infected with Wolbachia by parasitoids had increased survival and reduced development times. Overall, our study provides evidence for the horizontal transmission of Wolbachia between insect hosts by parasitic wasps, and the enhanced survival and reproductive abilities of insect hosts may adversely affect biological control programs.

Molecular cloning and characterization of a ß-1,3-glucan recognition protein from Plutella xylostella (L.).

The ß-glucan recognition proteins (ßGRPs) play a significant role as important pattern recognition proteins (PRPs) for recognizing conserved surface determinants of pathogens and trigger complex signaling pathways in invertebrates. In the present study, a full-length cDNA 1793bp encoding 479 amino acids and ßGRP1 was obtained from Plutella xylostella by reverse transcription PCR (RT-PCR) (designed as P×ßGRP1) which showed significant similarities with other insect's ßGRPs. The transcription level was constitutively expressed and upregulated by microbial induction in all life stages of P. xylostella. Tissue distribution showed P×ßGRP1 to be mainly expressed in fat body as detected by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Subsequent to knock down the P×ßGRP1 expression and the transcripts of Toll-like receptor, cecropin 1 and cecropin 2 decreased in P. xylostella. Meanwhile, the bacterial colonies increased and the expression of four AMP genes decreased on injection of anti-P×ßGRP1 into Bombyx mori. The results demonstrated that P×ßGRP1 can play a vital role in response to the expression of AMP genes in P. xylostella.

A new species and first record of the genus Cynegetis Chevrolat (Coleoptera, Coccinellidae, Epilachnini) from China.

The first species of the genus Cynegetis Chevrolat is recorded from China. Cynegetischinensis Wang & Ren, sp. n. is described from the Ningxia Province in North China. A key to the known species of Cynegetis is given. Diagnostic similarities and differences between Cynegetis and Subcoccinella Agassiz & Erichson are discussed and illustrated.

De novo sequencing-based transcriptome and digital gene expression analysis reveals insecticide resistance-relevant genes in Propylaea japonica (Thunberg) (Coleoptea: Coccinellidae).

The ladybird Propylaea japonica (Thunberg) is one of most important natural enemies of aphids in China. This species is threatened by the extensive use of insecticides but genomics-based information on the molecular mechanisms underlying insecticide resistance is limited. Hence, we analyzed the transcriptome and expression profile data of P. japonica in order to gain a deeper understanding of insecticide resistance in ladybirds. We performed de novo assembly of a transcriptome using Illumina's Solexa sequencing technology and short reads. A total of 27,243,552 reads were generated. These were assembled into 81,458 contigs and 33,647 unigenes (6,862 clusters and 26,785 singletons). Of the unigenes, 23,965 (71.22%) have putative homologues in the non-redundant (nr) protein database from NCBI, using BLASTX, with a cut-off E-value of 10(-5). We examined COG, GO and KEGG annotations to better understand the functions of these unigenes. Digital gene expression (DGE) libraries showed differences in gene expression profiles between two insecticide resistant strains. When compared with an insecticide susceptible profile, a total of 4,692 genes were significantly up- or down- regulated in a moderately resistant strain. Among these genes, 125 putative insecticide resistance genes were identified. To confirm the DGE results, 16 selected genes were validated using quantitative real time PCR (qRT-PCR). This study is the first to report genetic information on P. japonica and has greatly enriched the sequence data for ladybirds. The large number of gene sequences produced from the transcriptome and DGE sequencing will greatly improve our understanding of this important insect, at the molecular level, and could contribute to the in-depth research into insecticide resistance mechanisms.

RNAi-mediated knockdown of serine protease inhibitor genes increases the mortality of Plutella xylostella challenged by destruxin A.

Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides.

Extracellular lipase of an entomopathogenic fungus effecting larvae of a scale insect.

Lipases play an important role in the infection process of entomopathogenic fungi by hydrolyzing the ester bonds of lipoproteins, fats and waxes present on the insect surface and in the body. Here we report the purification and characterization of an extracellular lipase from Isaria fumosorosea. The enzyme was purified (138.46-fold) in three steps using (NH4 )2 SO4 precipitation followed by DEAE-cellulose and Sephadex G-100 column chromatography. The molecular weight of purified enzyme was determined to be 31 KDa by SDS-PAGE. The optimum temperature and pH for enzyme activity were 35 °C and 7.0, respectively, using p-nitrophenylpalmitate as the substrate. Lipolytic activity was enhanced in the presence of Ca(+2) , Mg(+2) , Na(+) , and NH4 (+) salts, while Zn(+2) , Fe(+2) , and Cu(+2) inhibited enzyme activity. The enzyme displayed broad substrate specificity with the highest activity observed for coconut oil and p-nitrophenyl carprate. Topical co-application of purified lipase with fungal conidial suspensions decreased the median survival time (ST50 ) of Dysmicoccus neobrevipes nymphs as compared to the fungus alone. Our results indicate that an extracellular lipase produced by I. fumosorosea can be exploited for development of enzyme-based insect management.

Host suitability comparison between the MEAM1 and AsiaII 1 cryptic species of Bemisia tabaci in cotton-growing zones of Pakistan.

BACKGROUND: Bemisia tabaci is a cryptic species complex. In Pakistan, members of the complex, MEAM1 and AsiaII 1, are the predominant species infesting cotton. The biology of the two on cotton, collard, cucumber and tomato was studied. RESULTS: In all cases there were significant interactions between species and host. MEAM1 developmental periods did not differ significantly across hosts, whereas AsiaII 1 developed more slowly on vegetables than on cotton. MEAM1 survival was highest on tomato (53.5 ± 1.1%), while AsiaII 1 survived best on cotton (67.3 ± 11.6%). MEAM1 longevity and fecundity were highest on tomato (14.7 ± 1.7 days and 82.4 ± 9.9 eggs), while AsiaII 1 longevity and fecundity were highest on cotton (23.7 ± 2.5 days and 135.2 ± 13.6 eggs). The MEAM1 intrinsic rates of increase (r(m)) on cotton and vegetable were similar (0.08-0.10), whereas the AsiaII 1 r(m) on cotton (0.15) was higher than on vegetables (0.11-0.13). The biology of MEAM1 from Pakistan was compared with published studies; it had a consistently slower rate of development, lower percentage survival, lower adult longevity, longer generation time, lower net reproductive rate and lower r(m). CONCLUSIONS: MEAM1 performed similarly across all hosts, whereas AsiaII 1 performed better on cotton. The comparison between the Pakistani MEAM1 with published studies suggests that the invasive MEAM1 may have higher performance.

Efficiency of plant induced volatiles in attracting Encarsia formosa and Serangium japonicum, two dominant natural enemies of whitefly Bemisia tabaci in China.

BACKGROUND: Whitefly Bemisia tabaci is a globally distributed and most destructive pest to agriculture. Owing to increasing chemical resistance, a long-lasting strategy to manage this pest must involve biological control. Herbivore-induced plant volatiles (HIPVs) usually play a profoundly important role in the foraging behaviour of natural enemies. Here, the effects of HIPVs from Chinese broccoli on the foraging behaviour of two dominant natural enemy species of B. tabaci in China, Encarsia formosa and Serangium japonicum, were investigated using a four-arm olfactometer, and exogenous jasmonic acid (JA) was used to induce plant volatiles to mimic the damage of the herbivore pest. RESULTS: The parasitoid E. formosa was found to be more attracted by the volatiles from JA-induced broccoli than those from control plants. The residence times of E. formosa in the final-choice areas closed to volatiles from shoot JA (SJA)- and root JA (RJA)-induced plants were 119.8 ± 35.2 s and 99.8 ± 34.7 s respectively in the dual-choice experiments, and 123.8 ± 32.0 s and 102.3 ± 28.7 s respectively in the three-choice experiment. All are significantly longer than those spent in the final-choice area closed to volatiles from control plants (CON). JA-induced volatiles were also only a little more attractive to the predator S. japonicum. However, there was no significant difference between each of the two natural enemies in residence time spent in the final-choice areas closed to SJA, RJA or CON volatiles in both the dual- and three-choice experiments. Furthermore, the number of times that the parasitoid or predator entered the SJA, RJA and CON final-choice areas was not significantly different in any of the experiments. CONCLUSION: The present results indicate that RJA- and SJA-induced plant volatiles have higher efficiencies in attracting the natural enemies of whitefly B. tabaci. The attractive efficiencies varied according to the plant volatiles that JA induced, and also depended on the natural enemy species. The parasitoid E. formosa seems to be more sensitive to and attracted by the induced volatiles than the predator S. japonicum.

Molecular characterization of a short peptidoglycan recognition protein (PGRP-S) from Asian corn borer (Ostrinia furnacalis) and its role in triggering proPO activity.

Peptidoglycan recognition proteins (PGRPs) are non-specific immune molecules of insects, and vertebrates etc., but are not present in plants and nematodes. In the current experiment, a PGRP DNA sequence (2,910 bp containing four exons) was identified from genomic DNA library of Asian corn borer, Ostrinia furnacalis, and a full-length cDNA programming PGRP was cloned (designed as OfPGRP-S) with an open reading frame of 579 bp, having 192 amino acid. This inferred amino acid sequence showed maximum similarity to known lepidopteran PGRPs. Quantitative real-time PCR investigation disclosed the level of mRNA of OfPGRP-S to be constitutively expressed in the whole developmental stages and with higher expression in the mature larvae. Even more the OfPGRP-S was mainly expressed in immune capable organs i.e., fat body and midgut, and was strongly induced by injecting gram-positive bacteria i.e., Staphylococus aureus. Recombinant protein OfPGRP-S could bind to S. aureus and Bacillus thuringiensis which enhance proPO activation in the presence of these microbes. The results indicated that OfPGRP-S is an inducible protein acting as a receptor-type PGRP for enhancing the proPO activation on exposure to bacteria.