Expression of Small RNA in Aphis gossypii and Its Potential Role in the Resistance Interaction with Melon

Background

The regulatory role of small RNAs (sRNAs) in various biological processes is an active area of investigation; however, there has been limited information available on the role of sRNAs in plant-insect interactions. This study was designed to identify sRNAs in cotton-melon aphid (Aphis gossypii) during the Vat-mediated resistance interaction with melon (Cucumis melo).

Methodology/Principal Findings

The role of miRNAs was investigated in response to aphid herbivory, during both resistant and susceptible interactions. sRNA libraries made from A. gossypii tissues feeding on Vat⁺ and Vat⁻ plants revealed an unexpected abundance of 27 nt long sRNA sequences in the aphids feeding on Vat⁺ plants. Eighty-one conserved microRNAs (miRNAs), twelve aphid-specific miRNAs, and nine novel candidate miRNAs were also identified. Plant miRNAs found in the aphid libraries were most likely ingested during phloem feeding. The presence of novel miRNAs was verified by qPCR experiments in both resistant Vat⁺ and susceptible Vat⁻ interactions. The comparative analyses revealed that novel miRNAs were differentially regulated during the resistant and susceptible interactions. Gene targets predicted for the miRNAs identified in this study by in silico analyses revealed their involvement in morphogenesis and anatomical structure determination, signal transduction pathways, cell differentiation and catabolic processes.

Conclusion/Significance

In this study, conserved and novel miRNAs were reported in A. gossypii. Deep sequencing data showed differences in the abundance of miRNAs and piRNA-like sequences in A. gossypii. Quantitative RT-PCR revealed that A. gossypii miRNAs were differentially regulated during resistant and susceptible interactions. Aphids can also ingest plant miRNAs during phloem feeding that are stable in the insect.     

Seasonal Phenology and Species Composition of the Aphid Fauna in a Northern Crop Production Area

Background

The species diversity of aphids and seasonal timing of their flight activity can have significant impacts on crop production, as aphid species differ in their ability to transmit plant viruses and flight timing affects virus epidemiology. The aim of the study was to characterise the species composition and phenology of aphid fauna in Finland in one of the northernmost intensive crop production areas of the world (latitude 64°).

Methodology/Principal Findings

Flight activity was monitored in four growing seasons (2007–010) using yellow pan traps (YPTs) placed in 4–8 seed potato fields and a Rothamsted suction trap. A total of 58,528 winged aphids were obtained, identified to 83 taxa based on morphology, and 34 species were additionally characterised by DNA barcoding. Seasonal flight activity patterns analysed based on YPT catch fell into three main phenology clusters. Monoecious taxa showed early or middle-season flight activity and belonged to species living on shrubs/trees or herbaceous plants, respectively. Heteroecious taxa occurred over the entire potato growing season (ca. 90 days). Abundance of aphids followed a clear 3-year cycle based on suction trap data covering a decade. Rhopalosiphum padi occurring at the end of the potato growing season was the most abundant species. The flight activity of Aphis fabae, the main vector of Potato virus Y in the region, and Aphis gossypii peaked in the beginning of potato growing season.

Conclusions/Significance

Detailed information was obtained on phenology of a large number aphid species, of which many are agriculturally important pests acting as vectors of plant viruses. Aphis gossypii is known as a pest in greenhouses, but our study shows that it occurs also in the field, even far in the north. The novel information on aphid phenology and ecology has wide implications for prospective pest management, particularly in light of climate change.     

Capacity assessment of <Emphasis Type="Italic">Myzus persicae,Aphis gossypii</Emphasis> and <Emphasis Type="Italic">Aphis spiraecola</Emphasis> (Hemiptera: Aphididae) to acquire and retain PVY<Superscript>NTN</Superscript> in Tunisia

The study was carried out to investigate the ability of three aphids, Myzus persicae, Aphis gossypii and Aphis spiraecola, to acquire and retain the Potato Virus Y (PVY) isolate, PVY^NTN. Tobacco plants, Nicotiana tabacum var. Xanthi, were used as test plant for the virus inoculation and aphid acquisition. The serological test double-antibody sandwich enzyme-linked immunosorbent assay was applied for virus detection on the test plants and aphids. Furthermore, virus retention by aphids was also assessed using a monoclonal anti-PVY^N. Although a duration of 2 min was enough for the virus acquisition, the three tested aphids showed different capacities to retain PVY^NTN. The retention of PVY^NTN was 3 h for M. persicae and A. spiraecola, and 2 h for A. gossypii. This study provides basic information of the virus retention by potato-colonizing aphid species, which may increase our understanding of PVY epidemiology in Tunisia.     

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.     

棉蚜抗氧化乐果品系及敏感品系羧酸酯酶性质的比较

在室内用氧化乐果逐代筛选的棉蚜抗性品系,相对于敏感品系的抗性倍数是17。用α-乙酸萘酯（α-NA）、α-丁酸萘酯（α-NB）、α-磷酸萘酯（α-NP）和β-磷酸萘酯（β-NP）作底物比较研究了氧化乐果抗性和敏感品系棉蚜Aphis gossypii羧酸酯酶的比活力、米氏常数（K_m）和最大反应速度V_max）等有关的动力学常数。以α-NA和α-NB作底物时,抗性品系棉蚜的比活力显著低于敏感品系的;以α-NP和β-NP作底物时,两个品系棉蚜的比活力、K_m和V_max没有明显差异。用α-NA、β-NA作底物染色做酯酶同工酶电泳,抗性品系棉蚜的酯酶同工酶染色比敏感品系棉蚜的浅。     

Symbiotic microbial studies in diverse populations of Aphis gossypii,existing on altered host plants in different localities during different times

Complex interactions between symbiotic bacteria and insects ultimately result in equilibrium in all aspects of life in natural insect populations. In this study, abundance of principal symbiotic bacteria was estimated using qPCR in 1553 individuals of aphids, Aphis gossypii. Aphids were sampled from primary and secondary host plants—hibiscus and cotton. Hibiscus aphids were collected from 24 different locations in April, September, and November, whereas cotton aphids were collected between 2015 and 2017 from areas with wide variations in climatic conditions. About 30%–45% aphids were recorded with the most dominant symbiont, Arsenophonus. The other symbionts were in low frequency, and about 7% of aphids were noted with Hamiltonella, Acinetobacter, and Microbacterium, and 3% of aphids were verified with Serratia and Pseudomonas. Aphids infected with Hamiltonella, Arsenophonus, and Serratia can influence Buchnera densities. Hamiltonella has positive interaction with densities of Arsenophonus and Serratia. Almost 100% coinfection of Hamiltonella and Arsenophonus was detected in Xinxiang aphids and 50% coinfection was reported in aphids from North China, while no coinfection was detected in Hainan aphids. These findings describe the prevalence pattern and richness of core community of symbiotic bacteria in naturally occurring populations of A. gossypii and provide new insights for the study of symbiotic bacteria.     

Host Generated siRNAs Attenuate Expression of Serine Protease Gene in Myzus persicae

Background

Sap sucking hemipteran aphids damage diverse crop species. Although delivery of ds-RNA or siRNA through microinjection/feeding has been demonstrated, the efficacy of host-mediated delivery of aphid-specific dsRNA in developing aphid resistance has been far from being elucidated.

Methodology/Principal Findings

Transgenic Arabidopsis expressing ds-RNA of Myzus persicae serine protease (MySP) was developed that triggered the generation of corresponding siRNAs amenable for delivery to the feeding aphids. M. persicae when fed on the transgenic plants for different time intervals under controlled growth conditions resulted in a significant attenuation of the expression of MySP and a commensurate decline in gut protease activity. Although the survivability of these aphids was not affected, there was a noticeable decline in their fecundity resulting in a significant reduction in parthenogenetic population.

Conclusions/Significance

The study highlighted the feasibility of developing host based RNAi-mediated resistance against hemipteran pest aphids.     

The microbial flora of Aphis gossypii: Patterns across host plants and geographical space

The cotton aphid, Aphis gossypii, has a worldwide distribution and causes damage to numerous economically important crops. The bacterial symbionts associated with cotton aphids, sampled mainly from malvaceous and cucurbitaceous plants within Japan and Australia, were characterised using molecular profiling approaches. The goal was to document the aphid symbionts present and determine if patterns of microbial diversity are consistent with the existence of host plant related cryptic species in A. gossypii. The bacterial profiles of the aphids are diverse and reflect local geography more than host plant use.     

Cytopathology, Mode of Aphid Transmission and Search for the Casual Agent of Cotton Bunchy Top Disease

The cytopathological effects of cotton bunchy top (CBT) disease and its mode of transmission by Aphis gossypii Glover (cotton aphid), were studied. CBT infection affected the leaf epidermal layer producing a loose, ruptured and rough surface morphology with many stomata closed and misshapen. Roots of CBT‐infected plants showed reduced growth, small knots and a dark brown appearance. A single aphid per plant was capable of transmitting CBT at 5%, whereas three aphids per plant transmitted CBT to 50% of the cotton seedlings and 20 aphids per plant transmitted the disease agent to 80% of the cotton seedlings. Aphis gossypii acquired CBT after a minimum acquisition access period of 5 min and transmitted the agent after a minimum inoculation access period of 1 h. Both alate and apterous aphids and nymph instars 2, 3 and 4 of A. gossypii transmitted CBT. This preliminary data suggest that A. gossypii transmits CBT in a semi‐persistent manner. Myzus persicae Sulz (green peach aphid) was unable to transmit CBT. A comprehensive attempt to isolate the CBT agent, using a range of virological techniques including double‐stranded RNA extraction, two‐dimensional gel electrophoresis for viroid, circular DNA test, nanovirus polymerase chain reaction (PCR), luteovirus PCR and enzyme‐linked immunosorbent assay, phytoplasma test, nucleoprotein purification and electron microscopy, was unsuccessful, raising the possibility that CBT may be caused by a unique new pathogen.     

Spatio-temporal variation of strawberry aphid populations and their parasitoids

Aphids are common herbivores in the strawberry crop that can reduce plant vigor and fruit quality and also transmit viruses. Aphid species prefer diverse plant organs, which represent particular habitats of different quality for aphids and for the development of natural enemies’ populations. Different habitat units (young leaves, mature leaves, buds, flowers) of strawberry were sampled fortnightly during all seasons. We identified seven aphid species, namely Chaetosiphon fragaefolii, Aphis gossypii, and Macrosiphum euphorbiae, the most abundant. During the autumn, C. fragaefolli and M. euphorbiae were scarce and A. gossypii was denser on mature leaves, while during summer M. euphorbiae was absent. During the winter, C. fragaefolii predominated on buds and young leaves, A. gossypii on flowers, and both species on mature leaves. During the spring, C. fragaefolii was even more abundant on buds, A. gossypii predominated on mature leaves, and the three species were equally abundant on flowers and young leaves. Parasitoids emerged from A. gossypii, M. euphorbiae and Myzus persicae, but not from C. fragaefolii. Three Aphidius and two Aphelinus species were recovered. All primary parasitoid species emerged from A. gossypii, and secondary parasitoids emerged only from this aphid. Aphis gossypii parasitism on mature leaves was markedly higher in winter and summer than in autumn and spring. Parasitism of A. gossypii was independent of its density, and the number of parasitized aphids was never higher than six. Our results contribute to define the most appropriate sample unit to estimate aphid density of different species and provide information about seasonal natural parasitism.     

Activation of ethylene‐related genes in response to aphid feeding on resistant and susceptible melon and tomato plants

The simple gaseous compound ethylene (ET) has long been recognized as a common component of plant responses to insect feeding and pathogen attack. However, it is presently uncertain whether it plays a role in host–plant resistance to piercing–sucking insects such as aphids. In these experiments, we investigated the expression of key ET‐associated genes in resistant and susceptible interactions in two model systems: the tomato‐Mi‐Macrosiphum euphorbiae (Thomas) (Hemiptera: Aphididae: Macrosiphini) system and the melon‐virus aphid transmission gene (Vat)‐Aphis gossypii Glover (Hemiptera: Aphididiae: Aphidini) system. We examined expression patterns of genes associated with ET synthesis, perception, signal transduction, and downstream response. When compared with control plants, plants infested with aphids showed marked differences in gene expression. In particular, ET signaling pathway genes and downstream response genes were highly upregulated in the resistant interaction between A. gossypii and Vat⁺, indicating ET may play a role in Vat‐mediated host–plant resistance. A key integrator between the ET and jasmonic acid pathways (Cm‐ERF1) showed the strongest response.     

Host instar suitability of Aphis gossypii (Hemiptera: Aphididae) for Lysiphlebus testaceipes (Hymenoptera: Braconidae) and parasitism effect on aphid life table

Lysiphlebus testaceipes (Cresson) (Hymenoptera: Braconidae, Aphidiinae) has constituted a well-studied parasitoid insect model, but very little is known about the host-instar suitability of aphid for the wasp so far. One of the hosts of L. testaceipes is Aphis gossypii Glover (Hemiptera: Aphididae). The latter is a serious aphid pest to vegetable production in Benin. Therefore, the objectives of our study were to: (1) examine the oviposition behavior of L. testaceipes on A. gossypii; (2) investigate the host-instar suitability of A. gossypii for L. testaceipes; and (3) compare the life table parameters of A. gossypii with aphids parasitized by L. testaceipes and unparasitized aphids (control). The study was conducted in a laboratory at 26 ± 1 °C in petri dishes and revealed that the parasitoid utilized up to seven stabbing stings to handle and oviposit, particularly in older A. gossypii. In aphids parasitized at the third instar, the net reproductive rate R _o as well as the intrinsic rate of natural increase r _m was significantly lower (2.119 ± 0.272 and 0.110 ± 0.018) compared to the control (15.529 ± 1.287 and 0.272 ± 0.008), respectively (p < 0.01).     

May the wild male loose? Male wing fanning performances and mating success in wild and mass-reared strains of the aphid parasitoid Aphidius colemani Viereck (Hymenoptera: Braconidae: Aphidiinae)

Aphidius colemani Viereck (Hymenoptera: Braconidae) is a pan-tropical broadly oligophagous endoparasitoid of many aphids of economic importance, including Aphis gossypii Glover and Myzus persicae (Sulzer). While the trophic interactions occurring among A. colemani and its hosts have been extensively studied, little is known about the male- and female-borne cues that guide mating dynamics. Male wing fanning has been found to play a key role in A. colemani courtship, as successful mounting of females without initial wing fanning has never been observed. In this research, we analyzed wing fanning performance and mating ability of males from three different strains of A. colemani: wasps commercially mass-reared on A. gossypii, wild wasps from parasitized A. gossypii, and wild wasps from parasitized Aphis nerii Boyer de Fonscolombe. Results showed that virgin females did not rely on particular male fanning features during mate choice. Moreover, when A. colemani individuals developed on A. gossypii, no major differences were detected in courtship and mating ability between field collected and mass-reared wasps. In contrast, courtship performance and mating success varied between wild A. colemani males reared on different hosts, with those developing on A. nerii having lower quality wing fanning performance during the mounting attempt phase, and reduced ability to compete for females with other males reared on A. gossypii.     

运用α-氰代酯荧光底物检测抗性棉蚜羧酸酯酶代谢活性

为了研究抗性和敏感棉蚜Aphis gossypii品系对菊酯类药剂代谢的差异, 本实验合成了溴氰菊酯和高效氯氰菊酯报告荧光底物, 应用这两种底物水解后生成具有荧光化合物的特性,测定了不同品系棉蚜羧酸酯酶的代谢活性。结果表明: 氧化乐果棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为10.0和3.4 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为4.0和2.4 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的2.9和1.7倍; 溴氰菊酯棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为7.6和6.2 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为9.3和5.2 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的1.2和1.8倍。这种衍生的报告荧光底物能够用来检测抗性棉蚜羧酸酯酶的水解活性, 表明羧酸酯酶可能参与棉蚜对溴氰菊酯和氧化乐果抗性的形成。     

Indirect multi-trophic interactions mediated by induced plant resistance: impact of caterpillar feeding on aphid parasitoids

Cotton produces insecticidal terpenoids that are induced by tissue-feeding herbivores. Damage by Heliothis virescens caterpillars increases the terpenoid content, which reduces the abundance of aphids. This effect is not evident in Bt-transgenic cotton, which is resistant to H. virescens. We determined whether induction of terpenoids by caterpillars influences the host quality of Aphis gossypii for the parasitoid Lysiphlebus testaceipes and whether this interaction is influenced by Bt cotton. The exposure of parasitoids to terpenoids was determined by quantifying terpenoids in the aphids. We detected several terpenoids in aphids and found a positive relationship between their concentrations in plants and aphids. When L. testaceipes was allowed to parasitize aphids on Bt and non-Bt cotton that was infested or uninfested with H. virescens, fewer parasitoid mummies were found on infested non-Bt than on Bt cotton. Important parasitoid life-table parameters, however, were not influenced by induced resistance following H. virescens infestation, or the Bt trait. Our study provides an example of a tritrophic indirect interaction web, where organisms are indirectly linked through changes in plant metabolites.     

Morphometric Relationship,Phylogenetic Correlation,and Character Evolution in the Species-Rich Genus Aphis (Hemiptera: Aphididae)

Background

The species-rich genus Aphis consists of more than 500 species, many of them host-specific on a wide range of plants, yet very similar in general appearance due to convergence toward particular morphological types. Most species have been historically clustered into four main phenotypic groups (gossypii, craccivora, fabae, and spiraecola groups). To confirm the morphological hypotheses between these groups and to examine the characteristics that determine them, multivariate morphometric analyses were performed using 28 characters measured/counted from 40 species. To infer whether the morphological relationships are correlated with the genetic relationships, we compared the morphometric dataset with a phylogeny reconstructed from the combined dataset of three mtDNA and one nuclear DNA regions.

Principal Findings

Based on a comparison of morphological and molecular datasets, we confirmed morphological reduction or regression in the gossypii group unlike in related groups. Most morphological characteristics of the gossypii group were less variable than for the other groups. Due to these, the gossypii group could be morphologically well separated from the craccivora, fabae, and spiraecola groups. In addition, the correlation of the rates of evolution between morphological and DNA datasets was highly significant in their diversification.

Conclusions

The morphological separation between the gossypii group and the other species-groups are congruent with their phylogenetic relationships. Analysis of trait evolution revealed that the morphological traits found to be significant based on the morphometric analyses were confidently correlated with the phylogeny. The dominant patterns of trait evolution resulting in increased rates of short branches and temporally later evolution are likely suitable for the modality of Aphis speciation because they have adapted species-specifically, rapidly, and more recently on many different host plants.     

RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius

Background

NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides.

Methodology/Principal Findings

The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs.

Conclusions/Significance

These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.