Aphid Megoura Viciae Research Articles

Functional characterization of alkaline phosphatases involved alarm pheromone in the vetch aphid Megoura viciae

The alkaline phosphatases (ALPs) are highly promiscuous enzymes and have been extensively investigated in mammals for their medical significance, but their functional promiscuity is relatively poorly understood in insects. Here, we first identified four ALP genes (designated as MvALP1-4) in the vetch aphid Megoura viciae that contained one alkaline phosphatase site, three metal-binding sites, and varied other functional sites. Phylogenetic analysis, molecular docking and the spatiotemporal expression profiling of MvALP1-4 were very different, indicating a promiscuous functionality. We also found that MvALP4 involved the biosynthesis of aphid alarm pheromones (EβF) invitro and invivo. Finally, transcriptome analysis in the stimulated and unstimulated aphids supported the involvement of MvALPs in the biosynthesis of aphid alarm pheromones. Our study identified a multifunctional ALP involved terpene synthase enzyme activity in the aphid, which contributes to the understanding of the functional plasticity of ALPs in insects.

Farnesyl/geranylgeranyl diphosphate synthases regulate the biosynthesis of alarm pheromone in a unique manner in the vetch aphid Megoura viciae.

Farnesyl/geranylgeranyl diphosphate synthases (FPPS/GGPPS) as the short-chain prenyltransferases catalyse the formation of the acyclic precursors (E)-FPP and (E)-GGPP for isoprenoid biosynthesis. Here, we first cloned the cDNAs encoding FPPS and GGPPS in the vetch aphid Megoura viciae (designated as MvFPPS and MvGGPPS). They had an open reading frame of 1185 and 930 bp in length, encoding 395 and 309 amino acids, with a theoretical isoelectric point of 6.52 and 6.21, respectively. Sequence alignment and phylogenetic analysis showed that MvFPPS and MvGGPPS shared the conserved aspartate-rich motifs characterized by all prenyltransferases identified to date and were clustered with their homologues in two large clades. RNA interference (RNAi) combined with gas chromatography/mass spectrometry (GC-MS) analysis showed that both MvFPPS and MvGGPPS were involved in the biosynthesis of alarm pheromone. Spatiotemporal expression profiling showed that the expression of MvFPPS and MvGGPPS was significantly higher in embryos than in other tissues. RNAi and GC-MS performed specifically in embryos corroborated the function of MvFPPS and MvGGPPS. In vitro, enzymatic activity assay and product analysis demonstrated that MvFPPS could catalysed the formation of (E)-FPP using DMAPP or (E)-GPP as the allylic cosubstrates in the presence of IPP, while MvGGPPS could only use (E)-GPP or (E)-FPP as cosubstrates. Functional interaction analysis using RNAi revealed that MvGGPPS exerts unidirectional functional compensation for MvFPPS. Moreover, it can regulate the biosynthesis of alarm pheromone by imposing a negative feedback regulation on MvFPPS. Our study helps to understand the molecular regulatory mechanism of terpenoid biosynthesis in the aphid.

Functional characterization of two different decaprenyl diphosphate synthases in the vetch aphid Megoura viciae.

Long-chain polyprenyl diphosphate synthases play a critical role in the formation of the prenyl side-chain of ubiquinones, but up to date, their functions have scarcely been characterized in insects. Here, we first cloned the complementary DNAs encoding the subunits of decaprenyl diphosphate synthase (DPPS) in the vetch aphid Megoura viciae, an important agricultural pest insect. The results showed that there existed three DPPS subunits, designated as MvDPPS1, MvDPPS2a, and MvDPPS2b, with an open reading frame of 1218, 1275, and 1290 bp, and a theoretical isoelectric point of 7.91, 6.63, and 9.62, respectively. The sequences of MvDPPS1s from different aphid species were nearly identical, while the sequences of MvDPPS2a and MvDPPS2b shared only moderate sequence similarity. Phylogenetic analysis clearly separated MvDPPS2a and MvDPPS2b, indicating a functional differentiation between them. Functional coexpression analysis in Escherichia coli showed that MvDPPS1 plus MvDPPS2a and MvDPPS1 plus MvDPPS2b, respectively, catalyzed the formation of the prenyl side-chain of the ubiquinone coenzyme Q10 (CoQ10). Interestingly, MvDPPS1 plus MvDPPS2b catalyzed the formation of the prenyl side-chain of a ubiquinone other than CoQ10. RNA interference-mediated knockdown of MvDPPS2a imposed no significant effect on MvDPPS2b, and vice versa, suggesting no compensatory action between them. In the end, we detected the product CoQ10 in the aphid, the first identification of CoQ10 in an insect species. Taken together, we characterized two functional DPPSs in M. viciae, one of which might be multifunctional. Our study helps to understand the functional plasticity of the terpenoid backbone biosynthesis pathway in insects.

Synthesis and Insecticidal Activity of β-D-Ribofuranoside Phosphate Derivatives

Phosphorylation of 4-methylphenyl 2,3-di-O-benzoyl-1-thio-β-D-ribofuranoside, followed by removal of the benzoyl protection afforded 1-S-(4-methylphenyl)-1-thio-β-D-ribofuranoside 5-phosphates. The synthesized compounds showed insecticidal activity against the vetch aphid Megoura viciae Buckt in combination with low toxicity.

Sensilla Morphology and Complex Expression Pattern of Odorant Binding Proteins in the Vetch Aphid Megoura viciae (Hemiptera: Aphididae).

Chemoreception in insects is mediated by several components interacting at different levels and including odorant-binding proteins (OBPs). Although recent studies demonstrate that the function of OBPs cannot be restricted to an exclusively olfactory role, and that OBPs have been found also in organs generally not related to chemoreception, their feature of binding molecules remains undisputed. Studying the vetch aphid Megoura viciae (Buckton), we used a transcriptomic approach to identify ten OBPs in the antennae and we examined the ultrastructural morphology of sensilla and their distribution on the antennae, legs, mouthparts and cauda of wingless and winged adults by scanning electron microscopy (SEM). Three types of sensilla, trichoid, coeloconic and placoid, differently localized and distributed on antennae, mouthparts, legs and cauda, were described. The expression analysis of the ten OBPs was performed by RT-qPCR in the antennae and other body parts of the wingless adults and at different developmental stages and morphs. Five of the ten OBPs (MvicOBP1, MvicOBP3, MvicOBP6, MvicOBP7, and MvicOBP8), whose antibodies were already available, were selected for experiments of whole-mount immunolocalization on antennae, mouthparts, cornicles and cauda of adult aphids. Most of the ten OBPs were more expressed in antennae than in other body parts; MvicOBP1, MvicOBP3, MvicOBP6, MvicOBP7 were also immunolocalized in the sensilla on the antennae, suggesting a possible involvement of these proteins in chemoreception. MvicOBP6, MvicOBP7, MvicOBP8, MvicOBP9 were highly expressed in the heads and three of them (MvicOBP6, MvicOBP7, MvicOBP8) were immunolocalized in the sensilla on the mouthparts, supporting the hypothesis that also mouthparts may be involved in chemoreception. MvicOBP2, MvicOBP3, MvicOBP5, MvicOBP8 were highly expressed in the cornicles-cauda and two of them (MvicOBP3, MvicOBP8) were immunolocalized in cornicles and in cauda, suggesting a possible new function not related to chemoreception. Moreover, the response of M. viciae to different components of the alarm pheromone was assessed by behavioral assays on wingless adult morph; (-)-α-pinene and (+)-limonene were found to be the components mainly eliciting an alarm response. Taken together, our results represent a road map for subsequent in-depth analyses of the OBPs involved in several physiological functions in M. viciae, including chemoreception.

Analysis of insect holocentric chromosomes by atomic force microscopy.

In order to go in depth into the analysis of holocentric chromosome structure, atomic force microscopy (AFM) was applied to metaphase plates of the aphid Megoura viciae. AFM showed that aphid chromatids adhere to one another without any prominent structure detectable between them and without any evidence of chromosomal constrictions. AFM thus provided new and reliable evidences at a nanomolecular level concerning the holocentric structure of aphid chromosomes, without any of the artefacts due to sample staining or coating that are usually associated with electron microscopy.

NOR heteromorphism within a parthenogenetic lineage of the aphid Megoura viciae.

In parthenogenetic females of a clone of the aphid Megoura viciae (Homoptera, Aphididae), more than 50% of the cells show heteromorphism between homologous NORs which are located on one telomeric region of the two X chromosomes. Using different techniques, such as staining with the CG-specific fluorochrome chromomycin A3, silver staining and in-situ hybridization with an rDNA probe, we have shown that the observed heteromorphism is due to an unequal distribution of ribosomal genes between homologous NOR regions. The total number of rDNA genes per individual aphid remained constant. Moreover, the analysis of cells from single embroys has shown that the observed heteromorphism is not only intraclonal but also intraindividual. These data, together with the finding of X chromosomes connected by chromatin bridges between their NORs, allow us to suggest that mitotic unequal crossing over could be the main cause of NOR heteomorphism in this taxon.

Olfactory antennal responses to plant volatiles in apterous virginoparae of the vetch aphid Megoura viciae

AbstractElectroantennogram (EAG) responses were recorded from apterous virginoparae of the vetch aphid Megoura viciae Buckton (Homoptera, Aphididae) to more than eighty volatile compounds in order to investigate its sensory ability to perceive plant odours. The response profile of M. viciae reveals a differential sensitivity for the array of plant volatiles tested. The whole group of general green leaf volatiles is very stimulatory. In addition to (E)‐2‐hexenal, the following compounds of this group elicit large EAG responses: (E)‐2‐heptenal, 1‐octenol‐3, hexyl acetate, (Z)‐3‐hexenyl acetate, hexanol‐1, hexanal, 2‐heptanone and 3‐octanone. Relatively large EAGs are also produced by 4‐methoxybenzaldehyde (p‐anisaldehyde), hexanonitrile, heptanonitrile, 1, 6‐hexanedithiol, butyl isothiocyanate, 4‐pentenyl isothiocyanate, (‐)‐(1S)‐β‐pinene, (+)‐(S)‐carvone, (‐)‐(R)‐carvone, α‐terpineol, linalool and citronellal. The nitriles are the most effective of all plant volatiles tested. Structure‐activity relationships occur in various groups of chemicals and members of the green leaf volatiles, benzaldehydes, isothiocyanates and monoterpenes are ranked accordingly. In the group of green leaf volatiles, aliphatic aldehydes are more stimulating than the corresponding alcohols. EAG responses to series of saturated aliphatic alcohols and aldehydes reveal that C6 and C7 compounds are the most stimulatory. Dose‐response curves show that the rank order of EAG response amplitudes hardly changes at lower dosages. It is concluded that M. viciae perceives general plant volatiles as well as more‐specific components, such as nitriles and isothiocyanates, associated with the odour blends of non‐host plant species.

The photoperiodic counter in the black bean aphid, Aphis fabae

The photoperiodic counter mechanism in the black bean aphid, Aphis fabae, was analysed by exposing newly-born, presumptive gynoparae to a variety of photoperiodic regimes at 15°C, and by the development of a model. It was shown that the counter accumulated long as well as short scotophases and that the “inductive strength”, or “value”, of long and short scotophases depended on photophase duration. Whilst the value of a long, 12-h, scotophase increased when accopanied by long photophases (>32 h), the value of a short, 8-h, scotophase decreased when accompanied by short photophases (<14 h). There was no detectable change in sensitivity to either long or short nights during the 4-day postnatal sensitive period. The counter mechanism appeared similar to that of the vetch aphid Megoura viciae, except that in M. viciae the value of a 12-h scotophase decreased with long photophases, whilst it increased in A. fabae.

Circadian rhythmicity is involved in photoperiodic time measurement in the aphidMegoura viciae

The photoperiodic clock in the vetch aphidMegoura viciae is generally accepted to be based on a non-circadian mechanism or “hourglass”, as no evidence has been found for the involvement of the circadian system in the photoperiodic response. By using a recently-devised protocol which discriminates between single and repeated night length measurement, we demonstrate here that long-night measurement inMegoura is executed in a repetitive way, and thus that its photoperiodic clock is based on a circadian oscillator after all. However, it is also apparent that the determination of short nights is not repetitive.

A model for the photoperiodic counter in the aphid Megoura viciae

The photoperiodic counter in the aphid Megoura viciae was analysed by means of a previously developed general model of the photoperiodic counter mechanism in insects and mites. It was shown that the counter in M. viciae accumulated long scotophases as well as short scotophases. It could further be shown that the inductive strength, or “value”, of long and short scotophases depended to a certain extent on photophase duration: long, 12-h scotophases accompanied by long photophases (> 18 h) decreased in value, whereas the inductive strength of short, 8-h scotophases decreased when accompanied by short photophases (< 16 h). Sensitivity to photoperiod changed slightly during the 10-day postnatal sensitive period at 15°C. Although sensitivity to short-night cycles (16 h light-8 h dark) showed a maximum at around day 7 and thereafter decreased sharply, sensitivity to long-night cycles (12 h light-12 h dark) did not appear to change. In M. viciae the “required day number”, which is the number of long- or short-night cycles required to elicit a 50% response, could only be determined at 12 and 15°C, as at higher temperatures (17 and 20°C) the long-night response (ovipara-production) was attenuated. For the low temperatures it was found that the accumulation of short-night cycles was far more temperature sensitive than that of long-night cycles. The observed attenuation of the long-night response at the higher temperatures could be explained in terms of the model, by assuming that the “minimally required induction sum” ( S min) is extremely sensitive to temperature, with a temperature coefficient much higher than the temperature coefficients of the photoperiodic values of both long-night cycles and short-night cycles.

Light-break experiments with emphasis on the quantitative perception of nightlength in the aphid Megoura viciae

The work is an attempt to link extensive studies, directed to elucidate the mode of operation of the photoperiodic clock, to those that have led to the conclusion that daylength is perceived quantitatively, and that quantitative time measurement is the only function of the photoperiodic clock. Experiments comprised (1) the widely used night-interruption technique, (2) a quantitative assessment of the intensities of action exerted by asymmetrical skeleton photoperiods so constructed and (3) a quantitative comparison between the skeleton and “ordinary” 24-h photoperiods. It was shown that the quantitative evaluation of photoperiod in M. viciae is based on the same fundamental process of nightlength measurement revealed in earlier studies on the qualitative photoperiodic response. This process appeared to be the same, notwithstanding the fact that long- or short-day photoperiods are perceived. It was inferred from the data presented that the dark process which participates in the act of nightlength measurement, consists of only two consecutive stages: the reversible starting phase and the irreversible main phase.

The photoperiodic counter, quantitative day-length effects and scotophase timing in the vetch aphid Megoura viciae

The development of parthenogenetic or sexual female forms (virginoparae and oviparae, respectively) in the aphid Megoura viciae is determined by the photoperiodic conditions perceived by the mother at any stage in her development from late embryo to young adult. A sensitive period for this response was selected using standard long (16 h light-8 h dark) and short days (12 h light–12 h dark) such that the form of the first ten progeny born was affected. At 12°C this sensitive period covered the first 11 days of maternal postnatal development, at 15°C it was 10 days while at higher temperatures (17 and 20°C) the short-day response (ovipara production) was attenuated. The required day number for a 50% response at 15°C was 3 days for long days but 7–8 days for short days indicating that long days are photoperiodically more potent. Experiments at 12 and 15°C showed that the accumulation of long-day information was more temperature sensitive than the accumulation of short-day information. The photoperiodic response of M. viciae depends primarily on the length of the dark period (scotophase). Novel experimental protocols and non-24 h light-dark cycles revealed that the photoperiodic counter accumulated long nights (short days) irrespective of photophase length. Short nights (long days), on the other hand, were not summated; moreover their effect depended on the length of the photophase. It appears that, although short nights are photoperiodically stronger, they serve only to modify the counter's accumulation of long nights. The quantitative effects (i.e. photoperiodic strength) of different 24-h light-dark cycles was investigated over experimental periods close to the required day number. As expected, photoperiodic strength decreased when scotophase lengths were close to critical (9.5 h). Long nights of 12–20 h were equally effective but short nights were maximally effective at 8 h. The four stages of dark-period measurement in the hour-glass clock were also reflected in these experiments.

Different Tolerance to High Temperatures Between Male and Female Embryos of the AphidMegoura viciae

Summary The total fecundity and pattern of reproduction were studied in the aphid Megoura viciae, under different postnatal temperature and photoperiod conditions, after prenatal exposure to long (16L:8D) or short (8L:16B) photoperiods. In the 13–27°C temperature range, total fecundity is markedly reduced by higher temperatures; postnatal photoperiod is without effect. In the temperature range 13–24°C, no difference was detected between aphids exposed prenatally to long and short photoperiods, respectively, whereas at 27°C fecundity of females exposed prenatally to short photoperiods is significantly lower. This reduced fecundity is due to the selective death of male embryos at a late developmental stage. The results provide a further case of low viability of the male sex in aphids.

Identification of an aphid sex pheromone

Although aphids reproduce asexually on their host plants during the summer, many species migrate to a winter host where sexual reproduction occurs. Males of certain species locate mates by means of a sex attractant pheromone released from the hind legs of the females1–4. Here we report the identification of such a pheromone from the vetch aphid Megoura viciae. Extracts of the excised legs of sexual females were analysed by gas chromatography-coupled single cell recording techniques (GC-SCR)5 and by gas chromatography-coupled mass spectrometry (GC-MS). A tentative identification by MS analysis of the two active components as specific isomers of the monoterpenoids nepetalactone (I), a known cat attractant, and nepetalactol (II) was confirmed by comparison with authenticated compounds isolated or synthesized from natural products. Although neither compound was attractive to males when presented alone in the laboratory bioassay, a mixture of the two components produced a response equal to that elicited by the female leg extract. Thus the sex pheromone of M. viciae comprises a synergistic mixture of the nepetalactone I and the nepetalactol II. The identification of aphid sex pheromones will provide a further means of investigating and manipulating the behaviour of these ubiquitous pests.

The behaviour and coupling of the photoreceptor and hour-glass photoperiod timer at low temperature in the aphid Megoura viciae

In Megoura viciae photoperiodic time is measured during the scotophase by an hour-glass mechanism which is temperature compensated within the range 6–20°C. At lower temperatures the clock “loses time”, running at about half the normal rate at 0°C. The present experiments, based on the analysis of critical night lengths, showed that for short periods of scotophase chilling (4h or less) the same diminished rate is maintained irrespective of the position of the low temperature period within the scotophase. This occurs despite the known changes in spectral sensitivity of the photoreceptor. With longer periods of chilling (>6h) a normal critical response was not observed unless several hours of darkness intervened between the end of the chilling period and the temperature-modified critical night length. This is interpreted as a “recovery” process involving the photoreceptor but not the hour-glass. Additional data indicated that the sequence of changes in the photoreceptor, including the early resetting phase, is closely coupled to the progression of the timer even when photosensitivity is temporarily impaired by low temperature.

Some effects of temperature on the hour glass photoperiod timer in the aphid Megoura viciae

“Resonance” experiments conducted at 3 temperatures do not support the view that the Megoura photoperiod (scotophase) timer has an oscillatory component. The main effects of temperature, which are consistent with an hour glass model, have been examined by exposing the aphids to temperatures ranging from −3 to 30°C for the first 4 h of the scotophase, subsequently returning them in each cycle to 15°C for measurement of the critical night length. During the temperature treatments the insects were removed from the plant to avoid indirect effects resulting from changes in the rate of development (this procedure causes a slight shift in the critical night length from 9.5 to 9.0 h). The timer was found to be almost perfectly compensated between 6 and 20°C, but at lower temperatures it runs slow, only one quarter the normal elapsed time being measured at −3°C. At higher temperatures (25 and 30°C) considerable irregularities appear, eventually resulting in the elimination of the critical night length. The results suggest that the timer may sometimes be stopped. Light perception was assessed by cooling the aphids in light. Photoreception was impaired in some individuals at temperatures as high as +6°C and became negligible at −3°C. The illuminated phase was then accepted as a dark interval of equal duration. Yet at this temperature the timer was still operating, albeit slowly. Their differing temperature thresholds point to some degree of separation in these two functions. High-temperature (25°C) experiments with continously feeding aphids showed that although scotophase heating was more effective, there was a distinct response when high temperatures were synchronized with the photophase. This was attributed to the operation of a “photoperiod counter” mechanism.

The Effect of Reproductive Investment on Pre-Reproductive Mortality in Aphids

(1) Ovariole number in the aphid Megoura viciae is a measure of the total reproductive investment during nymphal development. (2) Growth rate and ovariole number are not related. (3) On nutritionally poor hosts aphids with many ovarioles are less likely to survive to maturity than those with fewer ovarioles. (4) Pre-reproductive adults with few ovarioles are more resistant to starvation than those with many. (5) Pre-reproductive survival declines sigmoidally with increasing ovariole number.

Differential sensitivity of neurosecretory and non‐neurosecretory neurones of an aphid to sucrose during fixation for electron microscopy

SUMMARYThe Group 1 neurosecretory cells and a proportion of the non‐neurosecretory cells in the protocerebrum of the aphid Megoura viciae show gross differences in preservation when sucrose is used to increase the osmolarity of a glutaraldehyde primary fixative and an osmium tetroxide secondary fixative. The neurosecretory cells are well preserved whilst a proportion of the non‐neurosecretory cells are shrunken and electron dense, the proportion of affected cells increases with the concentration of sucrose used. This difference is not seen when either the primary or the secondary fixatives are used alone, when a mixture of the primary and secondary fixatives is used or when the osmotic pressure is increased with inorganic salts.

An electron microscopic study of flight muscle breakdown in an aphid Megoura viciae

Within two days of settling to feed on a host plant degenerative changes can be detected in the flight muscles of aphids at the ultrastructural level. The thick myosin filaments and the M Lines and later the Z bands disappear leaving the thin actin filaments in the cytoplasm. The mitochondria change their configuration and become enveloped in cytosegresomes. As mitochondria become reduced in number the numbers of lysosomes and residual bodies in the cells build up. The cells then appear to become dispersed as finger-like processes protrude from them and are pinched off.