Genome-wide identification of the expansin gene family reveals that expansin genes are involved in fibre cell growth in cotton.

BACKGROUND: Expansins (EXPs), a group of proteins that loosen plant cell walls and cellulosic materials, are involved in regulating cell growth and diverse developmental processes in plants. However, the biological functions of this gene family in cotton are still unknown. RESULTS: In this paper, we identified a total of 93 expansin genes in Gossypium hirsutum. These genes were classified into four subfamilies, including 67 GhEXPAs, 8 GhEXPBs, 6 GhEXLAs, and 12 GhEXLBs, and divided into 15 subgroups. The 93 expansin genes are distributed over 24 chromosomes, excluding Ghir_A02 and Ghir_D06. All GhEXP genes contain multiple exons, and each GhEXP protein has multiple conserved motifs. Transcript profiling and qPCR analysis revealed that the expansin genes have distinct expression patterns among different stages of cotton fibre development. Among them, 3 genes (GhEXPA4o, GhEXPA1A, and GhEXPA8h) were highly expressed in the initiation stage, 9 genes (GhEXPA4a, GhEXPA13a, GhEXPA4f, GhEXPA4q, GhEXPA8f, GhEXPA2, GhEXPA8g, GhEXPA8a, and GhEXPA4n) had high expression during the fast elongation stage, and GhEXLA1c and GhEXLA1f were preferentially expressed in the transition stage of fibre development. CONCLUSIONS: Our results provide a solid basis for further elucidation of the biological functions of expansin genes in relation to cotton fibre development and valuable genetic resources for future crop improvement.

[The effects of transgenic Cry1Ac+Cry2Ab cotton on cotton bollworm control and functional response of predators on whitefly].

In this study, we detected and clarified the roles of transgenic Cry1Ac+Cry2Ab cotton "639020" in controlling cotton bollworm (Helicoverpa armigera) during critical periods of bud stage (second generation of bollworm), flowering stage (third generation of bollworm) and bolling stage (fourth generation of bollworm) as well as the influences of 639020 cotton on functional response of the main predators (Chrysopa sinica larvae, Propylaea japonica, Orius and Erigonidium graminicola ) on whitefly using transgenic Cry1Ac cotton "CCRI41" and conventional cotton "CCRI49" as the control. Our results showed that the 639020 cotton well controlled the second and third generation of bollworm, and the level of insect resistance increased by 52.85% and 16.22% separately compared with that of CCRI41, with a significant effect on the second generation of bollworm. Moreover, the number of bollworm eggs in 639020 cotton field was lower than that in CCRI41 and CCRI49 cotton fields (except the second generation of bollworm) during the cotton bud, flowering and bolling stages. Although the number of bollworm larvae in 639020 cotton field was significantly lower than that in CCRI49 field, and both under the controlling index, it has no significant difference compared with that in CCRI41 cotton field. There were also no obvious changes in predator functions of Chrysopa sinica, Propylaea japonica, Orius and Erigonidium graminicola on bemisia tabaci between 639020, CCRI41 and CCRI49 cotton filed. This study evaluated the safety of new transgenic cotton on environment, anti-insect activity of exogenous gene and the safety of production and application prospect.

Identification and binding characterization of three odorant binding proteins and one chemosensory protein from Apolygus lucorum (Meyer-Dur).

For the sucking insect, Apolygus lucorum, taste is essential for finding host plants and oviposition sites. In A. lucorum, taste relies largely on the sensory system located within its proboscis. In this study, we constructed a cDNA library from A. lucorum proboscises and conducted preliminary analysis of 1554 ESTs. From this collection, we identified three putative odorant-binding proteins (AlucOBP3, AlucOBP4, AlucOBP6) and one chemosensory protein (AlucCSP1). Quantitative real-time polymerase chain reaction (qPCR) was used to study the expression pattern of these four genes. All four were expressed mainly in antennae, proboscises and legs, suggestive of roles in olfaction and gustation. We expressed and purified recombinant versions of AlucOBP3, AlucOBP4, AlucOBP6, and AlucCSP1 in a prokaryotic expression system. The ligand-binding specificities of the four proteins then were investigated in competition assays using 4,4'-dianilino-1,1'-binaphthyl-5, 5'-sulfonic acid (bis-ANS) as a probe. Of the 58 small organic compounds and five cotton secondary metabolites tested, plant volatiles cannot effectively displace bis-ANS from any of the four proteins. In contrast, most of the cotton secondary metabolites have high affinities for the three OBPs and AlucCSP1, indicating that these binding proteins more likely play a role in gustation than in olfaction.

Effects of Soil Salinity on the Expression of Bt Toxin (Cry1Ac) and the Control Efficiency of Helicoverpa armigera in Field-Grown Transgenic Bt Cotton.

An increasing area of transgenic Bacillus thuringiensis (Bt) cotton is being planted in saline-alkaline soil in China. The Bt protein level in transgenic cotton plants and its control efficiency can be affected by abiotic stress, including high temperature, water deficiency and other factors. However, how soil salinity affects the expression of Bt protein, thus influencing the control efficiency of Bt cotton against the cotton bollworm (CBW) Helicoverpa armigera (Hübner) in the field, is poorly understood. Our objective in the present study was to investigate the effects of soil salinity on the expression of Bt toxin (Cry1Ac) and the control efficiency of Helicoverpa armigera in field-grown transgenic Bt cotton using three natural saline levels (1.15 dS m-1 [low soil-salinity], 6.00 dS m-1 [medium soil-salinity] and 11.46 dS m-1 [high soil-salinity]). We found that the Bt protein content in the transgenic Bt cotton leaves and the insecticidal activity of Bt cotton against CBW decreased with the increasing soil salinity in laboratory experiments during the growing season. The Bt protein content of Bt cotton leaves in the laboratory were negatively correlated with the salinity level. The CBW populations were highest on the Bt cotton grown in medium-salinity soil instead of the high-salinity soil in field conditions. A possible mechanism may be that the relatively high-salinity soil changed the plant nutritional quality or other plant defensive traits. The results from this study may help to identify more appropriate practices to control CBW in Bt cotton fields with different soil salinity levels.

Phylogeographic patterns of Lygus pratensis (Hemiptera: Miridae): Evidence for weak genetic structure and recent expansion in northwest China.

Lygus pratensis (L.) is an important cotton pest in China, especially in the northwest region. Nymphs and adults cause serious quality and yield losses. However, the genetic structure and geographic distribution of L. pratensis is not well known. We analyzed genetic diversity, geographical structure, gene flow, and population dynamics of L. pratensis in northwest China using mitochondrial and nuclear sequence datasets to study phylogeographical patterns and demographic history. L. pratensis (n = 286) were collected at sites across an area spanning 2,180,000 km2, including the Xinjiang and Gansu-Ningxia regions. Populations in the two regions could be distinguished based on mitochondrial criteria but the overall genetic structure was weak. The nuclear dataset revealed a lack of diagnostic genetic structure across sample areas. Phylogenetic analysis indicated a lack of population level monophyly that may have been caused by incomplete lineage sorting. The Mantel test showed a significant correlation between genetic and geographic distances among the populations based on the mtDNA data. However the nuclear dataset did not show significant correlation. A high level of gene flow among populations was indicated by migration analysis; human activities may have also facilitated insect movement. The availability of irrigation water and ample cotton hosts makes the Xinjiang region well suited for L. pratensis reproduction. Bayesian skyline plot analysis, star-shaped network, and neutrality tests all indicated that L. pratensis has experienced recent population expansion. Climatic changes and extensive areas occupied by host plants have led to population expansion of L. pratensis. In conclusion, the present distribution and phylogeographic pattern of L. pratensis was influenced by climate, human activities, and availability of plant hosts.

Bacterial communities of the cotton aphid Aphis gossypii associated with Bt cotton in northern China.

Aphids are infected with a wide variety of endosymbionts that can confer ecologically relevant traits. However, the bacterial communities of most aphid species are still poorly characterized. This study investigated the bacterial diversity of the cotton aphid Aphis gossypii associated with Bt cotton in northern China by targeting the V4 region of the 16S rDNA using the Illumina MiSeq platform. Our sequencing data revealed that bacterial communities of A. gossypii were generally dominated by the primary symbiont Buchnera, together with the facultative symbionts Arsenophonus and Hamiltonella. To our knowledge, this is the first report documenting the facultative symbiont Hamiltonella in A. gossypii. Moreover, the bacterial community structure was similar within aphids from the same province, but distinct among those from different provinces. The taxonomic diversity of the bacterial community is greater in Hebei Province compared with in samples from Henan and Shandong Provinces. The selection pressure exerted by the different geographical locations could explain the differences found among the various provinces. These findings broaden our understanding of the interactions among aphids, endosymbionts and their environments, and provide clues to develop potential biocontrol techniques against this cotton aphid.

The Distribution and Host Shifts of Cotton-Melon Aphids in Northern China.

Aphis gossypii Glover (Hemiptera: Aphididae) is a serious pest of cotton in northern China. A microsatellite analysis was used to characterize the genetic structure of A. gossypii populations from different geographic, host plant, and seasonal populations in 2014. Among 906 individuals, 507 multilocus genotypes were identified, with genotypic richness values of 0.07-1.00 for the populations. We observed moderate levels of genetic differentiation among geographic populations (FST = 0.103; 95% confidence interval: 0.065-0.145) and host plant populations (FST = 0.237; 95% confidence interval: 0.187-0.296). A Mantel test of isolation by distance revealed no significant correlations between Slatkin's linearized FST and the natural logarithm of geographic distance. A Bayesian analysis of population genetic structures identified three clusters. An analysis of molecular variance revealed significant differences among the three clusters (F = 0.26596, P < 0.0001), among seasons (F = 0.04244, P = 0.00381), and among host populations (F = 0.12975, P = 0.0029). Thus, the A. gossypii populations in northern China exhibit considerable genotypic diversity. Additionally, our findings indicated that the 31 analyzed populations could be classified as one of three host biotypes (i.e., cotton, cucumber, and pomegranate biotypes). There were also clear seasonal effects on population genetic structure diversity among aphids collected from Anyang.

Bt proteins Cry1Ah and Cry2Ab do not affect cotton aphid Aphis gossypii and ladybeetle Propylea japonica.

Plant varieties expressing the Bt (Bacillus thuringiensis) insecticidal proteins Cry1Ah and Cry2Ab have potential commercialization prospects in China. However, their potential effects on non-target arthropods (NTAs) remain uncharacterized. The cotton aphid Aphis gossypii is a worldwide pest that damages various important crops. The ladybeetle Propylea japonica is a common and abundant natural enemy in many cropping systems in East Asia. In the present study, the effects of Cry1Ah and Cry2Ab proteins on A. gossypii and P. japonica were assessed from three aspects. First, neither of the Cry proteins affected the growth or developmental characteristics of the two test insects. Second, the expression levels of the detoxification-related genes of the two test insects did not change significantly in either Cry protein treatment. Third, neither of the Cry proteins had a favourable effect on the expression of genes associated with the amino acid metabolism of A. gossypii and the nutrition utilization of P. japonica. In conclusion, the Cry1Ah and Cry2Ab proteins do not appear to affect the cotton aphid A. gossypii or the ladybeetle P. japonica.

Identification of Aphis gossypii Glover (Hemiptera: Aphididae) Biotypes from Different Host Plants in North China.

BACKGROUND: The cotton-melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a polyphagous species with a worldwide distribution and a variety of biotypes. North China is a traditional agricultural area with abundant winter and summer hosts of A. gossypii. While the life cycles of A. gossypii on different plants have been well studied, those of the biotypes of North China are still unclear. RESULTS: Host transfer experiments showed that A. gossypii from North China has two host-specialized biotypes: cotton and cucumber. Based on complete mitochondrial sequences, we identified a molecular marker with five single-nucleotide polymorphisms to distinguish the biotypes. Using this marker, a large-scale study of biotypes on primary winter and summer hosts was conducted. All A. gossypii collected from three primary hosts-hibiscus, pomegranate, and Chinese prickly ash-were cotton biotypes, with more cotton-melon aphids found on hibiscus than the other two species. In May, alate cotton and cucumber biotypes coexisted on cotton and cucumber seedlings, but each preferred its natal host. Both biotypes existed on zucchini, although the cucumber biotype was more numerous. Aphids on muskmelon were all cucumber biotypes, whereas most aphids on kidney bean were cotton biotypes. Aphids on seedlings of potato and cowpea belong to other species. In August, aphids on cotton and cucumber were the respective biotypes, with zucchini still hosting both biotypes as before. Thus, the biotypes had different fitnesses on different host plants. CONCLUSIONS: Two host-specialized biotypes (cotton and cucumber) are present in North China. Hibiscus, pomegranate, and Chinese prickly ash can serve as winter hosts for the cotton biotype but not the cucumber biotype in North China. The fitnesses of the two host-specialized biotypes differ on various summer hosts. When alate aphids migrate to summer hosts, they cannot accurately land on the corresponding plant.

The complete mitochondrial genome of the Chrysoperla sinica (Tjeder).

The complete mitochondrial genome of the Chrysoperla sinica was sequenced in this study. It is 16,057 bp in length, and contains 22 transfer RNA genes, 13 protein-coding genes (PCGs), 2 rRNA genes and 1 non-coding AT-rich region. But, C. sinica lack long tandem repeats. The Chrysoperla sinica mitogenome displays a very high level of similarity (99.27%) with Chrysoperla nipponensis. The genome base composition is 39.24% A, 39.70% T, 11.98% C, and 9.08% G. Similar to other insects, all PCGs are initiated by a typical ATN codon, except COX1, which starts with TCG. Nine PCGs have the standard, complete stop codon (TAA or TAG), and the other four PCGs end with the incomplete stop codon (TA or T).

Identification and expression pattern of candidate olfactory genes in Chrysoperla sinica by antennal transcriptome analysis.

Chrysoperla sinica is one of the most prominent natural enemies of many agricultural pests. Host seeking in insects is strongly mediated by olfaction. Understanding the sophisticated olfactory system of insect antennae is crucial for studying the physiological bases of olfaction and could also help enhance the effectiveness of C. sinica in biological control. Obtaining olfactory genes is a research priority for investigating the olfactory system in this species. However, no olfaction sequence information is available for C. sinica. Consequently, we sequenced female- and male-antennae transcriptome of C. sinica. Many candidate chemosensory genes were identified, including 12 odorant-binding proteins (OBPs), 19 chemosensory proteins (CSPs), 37 odorant receptors (ORs), and 64 ionotropic receptors from C. sinica. The expression patterns of 12 OBPs, 19 CSPs and 37 ORs were determined by RT-PCR, and demonstrated antennae-dominantly expression of most OBP and OR genes. Our finding provided large scale genes for further investigation on the olfactory system of C. sinica at the molecular level.

Transcriptome comparison of the sex pheromone glands from two sibling Helicoverpa species with opposite sex pheromone components.

Differences in sex pheromone component can lead to reproductive isolation. The sibling noctuid species, Helicoverpa armigera and Helicoverpa assulta, share the same two sex pheromone components, Z9-16:Ald and Z11-16:Ald, but in opposite ratios, providing an typical example of such reproductive isolation. To investigate how the ratios of the pheromone components are differently regulated in the two species, we sequenced cDNA libraries from the pheromone glands of H. armigera and H. assulta. After assembly and annotation, we identified 108 and 93 transcripts putatively involved in pheromone biosynthesis, transport, and degradation in H. armigera and H. assulta, respectively. Semi-quantitative RT-PCR, qRT-PCR, phylogenetic, and mRNA abundance analyses suggested that some of these transcripts involved in the sex pheromone biosynthesis pathways perform. Based on these results, we postulate that the regulation of desaturases, KPSE and LPAQ, might be key factor regulating the opposite component ratios in the two sibling moths. In addition, our study has yielded large-scale sequence information for further studies and can be used to identify potential targets for the bio-control of these species by disrupting their sexual communication.

Functional characterizations of one odorant binding protein and three chemosensory proteins from Apolygus lucorum (Meyer-Dur) (Hemiptera: Miridae) legs.

Chemoreception plays an important role in insects for sensing information when searching for host and oviposition sites. An understanding of the chemosensory mechanism could aid in the development of new methods to effectively prevent damage from insects in agriculture. We have constructed a legs cDNA library for Apolygus lucorum and sequenced 1584 ESTs, from which we identified 669 unigenes. From this collection we identified one putative odorant binding protein (AlucOBP5) and three chemosensory proteins (AlucCSP2, AlucCSP3, AlucCSP4) genes. Using real-time PCR method, we assessed the expression of these genes in the head, thorax, abdomen, wing, antenna and mouthparts. Results indicate that the expression of these genes had tissue- and gender-specificity. AlucCSP2 and AlucCSP3 were specifically expressed in female wings. AlucCSP4 was expressed relatively highly in female wings but also expressed in other tissues. AlucOBP5 was expressed in female abdomen and male legs with high levels in the latter. Expression vectors for these proteins were constructed and expressed in BL21(DE3). The purified proteins were then tested for binding properties using bis-ANS as the fluorescent ligand. AlucOBP5 could bind strongly with phenyl acetaldehyde, 1-hexanol, 3-hexenal and ß-ionone. AlucCSP2 and AlucCSP3 had low affinity with all general odorants. AlucCSP4 did not bind with any of the standards. All four proteins could bind with gossypol, meletin with high affinity and could also bind with rutin hydrate, although AlucCSP4 had weak binding capacity. AlucCSP3 and AlucCSP4 could bind weakly with catechin, while AlucCSP2 and AlucOBP5 could not.

First Transcriptome and Digital Gene Expression Analysis in Neuroptera with an Emphasis on Chemoreception Genes in Chrysopa pallens (Rambur).

BACKGROUND: Chrysopa pallens (Rambur) are the most important natural enemies and predators of various agricultural pests. Understanding the sophisticated olfactory system in insect antennae is crucial for studying the physiological bases of olfaction and also could lead to effective applications of C. pallens in integrated pest management. However no transcriptome information is available for Neuroptera, and sequence data for C. pallens are scarce, so obtaining more sequence data is a priority for researchers on this species. RESULTS: To facilitate identifying sets of genes involved in olfaction, a normalized transcriptome of C. pallens was sequenced. A total of 104,603 contigs were obtained and assembled into 10,662 clusters and 39,734 singletons; 20,524 were annotated based on BLASTX analyses. A large number of candidate chemosensory genes were identified, including 14 odorant-binding proteins (OBPs), 22 chemosensory proteins (CSPs), 16 ionotropic receptors, 14 odorant receptors, and genes potentially involved in olfactory modulation. To better understand the OBPs, CSPs and cytochrome P450s, phylogenetic trees were constructed. In addition, 10 digital gene expression libraries of different tissues were constructed and gene expression profiles were compared among different tissues in males and females. CONCLUSIONS: Our results provide a basis for exploring the mechanisms of chemoreception in C. pallens, as well as other insects. The evolutionary analyses in our study provide new insights into the differentiation and evolution of insect OBPs and CSPs. Our study provided large-scale sequence information for further studies in C. pallens.

Ecological adaption analysis of the cotton aphid (Aphis gossypii) in different phenotypes by transcriptome comparison.

BACKGROUND: The cotton aphid, Aphis gossypii Glover, is a destructive insect pest worldwide; it directly or indirectly damages (virus transmission) 300 species of host plants. Knowledge of their ecologically adaptive mechanisms at the molecular level may provide an essential and urgent method to effectively control this pest. However, no transcriptome information is available for the cotton aphid and sequence data are scarce. Therefore, we obtained transcriptome data. RESULTS: To facilitate such a study, two cotton aphid transcriptomes at different growth stages of cotton, seedling and summer, were sequenced. A total of 161,396 and 66,668 contigs were obtained and assembled into 83,671 and 42,438 transcripts, respectively. After combining the raw date for both transcriptomes, the sequences were reassembled into 66,695 transcripts, and 52,160 were annotated based on BLASTX analyses. Comparison of the transcriptomes revealed that summer presented less challenges for the cotton aphids than the seedling stage of cotton. In total, 58 putative heat shock protein genes and 66 candidate cytochrome p450 genes were identified with BLASTX. CONCLUSIONS: Our results form a basis for exploring the molecular mechanisms of ecological adaption in the cotton aphid. Our study also provides a baseline for the exploration of abiotic stress responses. In addition, it provides large-scale sequence information for further studies on this species.