Facultative 'secondary' bacterial symbionts and the nutrition of the pea aphid, Acyrthosiphon pisum

Abstract.  Facultative 'secondary' bacterial symbionts influence various traits of aphids, including plant utilization patterns and resistance to parasitoids. The present study is designed to test the hypothesis that these multiple effects are underlain by symbiont-mediated changes to the aphid requirement for the dominant dietary nutrients, sucrose and amino acids. The performance of pea aphids ( Acyrthosiphon pisum ) on chemically defined diets of systematically altered sucrose and amino acid content varies among eight parthenogenetic clones, with a pattern that does not match the aphid complements of secondary symbionts, Hamiltonella defensa , Regiella insecticola and Serratia symbiotica . Aphid performance is reduced, increased and unaffected by elimination of S. symbiotica , R. insecticola and H. defensa , respectively, but with no significant effect on the range of diets on which aphids performed well. It is concluded that the impact of secondary symbionts on aphid traits is most unlikely to have a purely nutritional basis.     

Cannibalism in the pea aphid,Acyrthosiphon pisum

Previous observations of cannibalism have been made in the aphid Acyrthosiphon pisum （L.）： this article seeks to quantify factors contributing to such be- haviors. We observed and quantified the responses of a number of clones and life stages to varying levels of starvation, in the form of increasingly desiccated Vicafaba L. plants （receiving 50, 25, or 10 mL every second day） or a complete absence of host plant. We found that, while the longest incidences of cannibalism are carried out by juveniles （F = 3.45, P = 0.019, df = 3） and targeted at adults, the starvation treatments had the most significant effect on the prevalence of cannibalism in mature A. pisum （F ---- 2.24, P = 0.025, df = 9）. Furthermore, there was no difference between the prevalence or dura- tion of cannibalistic activities within and between different clones （P 〉 0.05 in all cases）, though juveniles were more likely to target unrelated aphids （V = 6 112, P = 0.011）, and spent more time feeding on aphids from the same culture （V = 6 062, P = 0.018）.     

Field estimation of variation in host plant use between local populations of pea aphids from two crops

Abstract. 1. In a reciprocal transplant experiment on pea aphids (Acyrthosiphon pisum (Harris)), the relative performance of clones collected from nearby alfalfa and red clover fields was tested by allowing clonal replicates to develop on both crops under field conditions.
2. Populations from alfalfa and red clover differed in relative survivorship and probabilities of reproduction on the two crops. Clones had significantly higher performance on the crop from which they were collected (the 'home' crop) than they did on the other crop.
3. Evidence is presented that previous experience on these host plants cannot account for the increased probability of reproduction observed on the 'home' crop. Thus, the differences between these two populations in their relative performance on alfalfa and clover are likely to be genetically based.
4. These results illustrate that local adaptation to different host plants can occur within small geographical areas, despite the high probability that migrants are exchanged between nearby fields of the two crops.
5. Experimental designs of the type described here permit estimation of patterns of genetic variation within and between insect populations. When applied to pest species, such designs will facilitate the study of evolution in agricultural systems.     

Effects of the maternal and pre‐adult host plant on adult performance and preference in the pea aphid,Acyrthosiphon pisum

Abstract 1. The taxon known as the pea aphid, Acyrthosiphon pisum, is composed of a series of host plant associated populations and is widely used as a model system to explore ecological speciation and the evolution of specialisation. It is thus important to know how maternal and pre‐adult experience influences host plant utilisation in this species. 2. The relative importance of the maternal and pre‐adult host plant for adult fecundity and host preference was investigated using three aphid clones collected from Lathyrus pratensis and maintained on Lathyrus or Vicia faba. 3. No significant effects of the maternal host plant on offspring fecundity were detected. 4. The host plant on which the aphid grew up influenced adult fecundity, although in a complex way that depended on both the adult host plant species and when after transfer to the test plant fecundity was assessed. 5. All three clones preferred to colonise Lathyrus over Vicia, and this preference was stronger for aphids raised on Lathyrus. 6. The significance of the results for studies of the evolution of specialisation and speciation that employ A. pisum is discussed.     

The photoperiodic responses and phenology of an English strain of the pea aphid Acyrthosiphon pisum

Abstract. 1. The response curves for the photoperiodic induction of the sexual forms (oviparae and males) differ significantly in an English clone of the pea aphid Acyrthosiphon pisum Harris. Male production is sharply peaked. The late summer scotophases (dark periods) that initiate male production are 1.0–1.5 h shorter than those that initiate ovipara production; the induction of males, but not oviparae, virtually ceases at scotophases longer than 12 h. This disparity suggests that there are two photoperiodic clocks.
2. All ovipara-producing aphids switch spontaneously to virginopara production part-way through the progeny sequence, irrespective of photoperiod. This may confer the ability to overwinter partheno-genetically under favourable climatic conditions. Since males are always born last in the progeny sequence this possibility would be pre-empted unless the terminal male sequences were suppressed by long winter scotophases.
3. The role of the photoperiodic response in determining the seasonal phenology was tested in the field by exposing a succession of laboratory-reared aphids to natural photoperiods, in late summer and autumn. Adult males and oviparae developed synchronously in early October, the difference in critical scotophase compensating for the late appearance of males in the progeny sequence. A large part of the 'civil twilight' is photoperiodically active.
4. Clones propagated outdoors by isolating aphids in each generation from the terminal sequence of virginoparae did not survive the whole winter of 1985/86. The chances of survival were reduced by severe weather and by the inability of the photoperiodic system to 'turn off' the production of early born oviparae which are, therefore, 'wasted'.     

Diversity and geographic distribution of secondary endosymbiotic bacteria in natural populations of the pea aphid,Acyrthosiphon pisum

In addition to the essential intracellular symbiotic bacterium Buchnera, several facultative endosymbiotic bacteria called collectively secondary symbionts (S-symbionts) have been identified from the pea aphid Acyrthosiphon pisum. We conducted an extensive and systematic survey of S-symbionts in Japanese local populations of A. pisum using a specific PCR detection technique. Five S-symbionts of A. pisum, PASS, PAUS, PABS, Rickettsia and Spiroplasma, and two facultative endosymbionts universally found in various insects, Wolbachia and Arsenophonus, were targeted. Of 119 isofemale strains originating from 81 localities, 66.4% of the strains possessed either of four S-symbionts: PASS (38.7%); PAUS (16.0%); Rickettsia (8.4%); and Spiroplasma (3.4%), while 33.6% of the strains contained only Buchnera. PABS, Wolbachia and Arsenophonus were not detected from the Japanese strains of A. pisum. In order to understand intra- and interpopulational diversity of S-symbiont microbiota in detail, 858 insects collected from 43 localities were examined for infection with the four S-symbionts. It was demonstrated that different S-symbionts coexist commonly in the same local populations, but double infections with two S-symbionts were rarely detected. Notably, the S-symbionts exhibited characteristic geographical distribution patterns: PASS at high frequencies all over Japan; PAUS at high frequencies mainly in the northeastern part of Japan; and Rickettsia and Spiroplasma at low frequencies sporadically in the southwestern part of Japan. These results indicate that the geographical distribution and infection frequency of the S-symbionts, in particular PAUS, might be affected by environmental and/or historical factors. Statistical analyses suggested that the distribution of PAUS infection might be related to host plant species, temperature and precipitation.     

The significance of a facultative bacterium to natural populations of the pea aphid Acyrthosiphon pisum

Abstract. 1.  Laboratory studies have implicated various accessory bacteria of aphids as important determinants of aphid performance, especially on certain plant species and under certain thermal regimes. One of these accessory bacteria is PABS (also known as T-type), which is distributed widely but is not universal in natural populations of the pea aphid Acyrthosiphon pisum in the U.K.
2.  To explore the impact of PABS on the performance of A. pisum , the nymphal development time and fecundity of aphids collected directly from natural populations and caged on the host plant Vicia faba in the field were quantified. Over 4 consecutive months June–September 1999, the performance of PABS-positive and PABS-negative aphids did not differ significantly.
3.  Deterministic modelling of the performance data showed that the variation in simulated population increase of PABS-positive and PABS-negative aphids would overlap substantially.
4.  Analysis of aphids colonising five host plants ( Lathyrus odoratus , Medicago sativa , Pisum sativum , Trifolium pratense , Vicia faba ) between April and September 2000 and 2001, identified no robust differences between the distribution of PABS-positive and PABS-negative aphids on different plants and with season or temperature.
5.  It is concluded that PABS is not an important factor shaping the performance or plant range of A. pisum under the field conditions tested. Reasons for the discrepancies between this study and laboratory-based studies are considered.     

High variation in host adaptation among clones of the pea aphid,Acyrthosiphon pisum on peas,Pisum sativum

The performance of one clone of the pea aphid,Acyrthosiphon pisum (Harris), was assessed on 37 different cultivars and species ofPisum L. In addition, random samples of 36 pea aphid clones collected on alfalfa and clover were tested on a selection of fivePisum sativum L. cultivars. Aphid performance was evaluated in terms of the mean relative growth rate (MRGR) during the first five days of life or other life history variables. The MRGR of the first-mentioned pea aphid clone differed little between cultivars. No significant differences in MRGR were found between wild and cultivatedPisum species or between modern and oldP. sativum cultivars. There was considerable variation in host adaptation among the 36 pea aphid clones within each sampled field. The pea aphid clones showed no consistent pattern in performance on four of the five pea cultivars i.e. there was a significant pea aphid genotype —pea genotype interaction. On one of the cultivars all clones performed well. Pea aphid clones collected from red clover generally performed relatively poorly on pea cultivars, in contrast to the pea aphid clones collected on alfalfa. There was no difference in performance between the two pea aphid colour forms tested. Possible reasons for the high variation and the observed adaptation patterns are discussed. The fact that all clones were collected in two adjacent fields indicates thatA. pisum shows high local intraspecific variability in terms of host adaptation.     

Identification and functional characterization of an odorant receptor in pea aphid,Acyrthosiphon pisum

The sensitive olfactory system is necessary for survival of insects.Odorant receptors (ORs)are located on the dendrites of olfactory receptor neurons and play a critical role in odor detection.Insect ORs are functionally analyzed via heterologous expression in a Xenopus oocyte system using a two-electrode voltage-clamp (TEVC)electrophysiological recording.Here,we have identified a novel OR in the pea aphid,Acyrthosiphon pisum,then we cloned and named it ApisOR4.We analyzed the ApisOR4 tissue expression patterns and found expression only in antennae tissues.Further functional analysis using TEVC revealed that ApisOR4 is broadly tuned to eight volatiles,which elicit electrophysiological response in pea aphid antennae.This study provides an initial functional analysis of aphid ORs and identifies candidate volatiles to be used in developing new strategies for aphid control.     

Microsatellite DNA markers for the pea aphid Acyrthosiphon pisum

Microsatellite loci were isolated from enriched partial genomic libraries of Acyrthosiphon loti and Acyrthosiphon pisum. Twenty of those loci were characterized in A. pisum. Fifteen of those loci were polymorphic. Genetic diversity varied across loci, allele repeat number ranging from two to 15, and observed heterozygosity from 0.1 and 0.96. An additional eight microsatellite loci originally isolated from other aphids but cross‐priming with A. pisum showed polymorphism as well. Allele size ranged from three to 9 and observed heterozygosity from 0.43 to 0.84. Overall, we present 23 microsatellite loci that can be used to reveal polymorphism in pea aphids.     

Juvenile hormone effects on polymorphism in the pea aphid, Acyrthosiphon pisum

When reared in short days (LD 12:12) at 15°C, apterous Acyrthosiphon pisum gave birth to sexual females (oviparae) exclusively for the first eight days of larviposition. After this time they switched to the production of parthenogenetic females (viviparae). Topical application of juvenile hormones I, II and III to fourth instar or adult ovipara-producers induced the precocious appearance of parthenogenetic females in the progeny sequence. Various forms intermediate between oviparae and viviparae were also produced and repetitive JH-I treatments resulted in a few alatiform progeny. However, many of the JH induced apterous, parthenogenetic females appeared to be normal viviparae and were capble of reproduction. Thus, prenatal treatment of oviparous embryos with JH diverts development towards the viviparous form. JH-I treatment of long-day reared A. pisum had no effect on the type of progeny produced.

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Effets de l'hormone juvénile sur le polymorphisme d'Acythosiphon pisum

Résumé Quand il est élevé sous jours courts (LN 12/12) à 15°C, le type anglais vert d'Acyrthosiphon pisum ne donne naissance qu'à des femelles sexuées (ovipares) pendant la première partie de sa période de reproduction. Ensuite quelques types intermédiaires ovipares/vivipares peuvent apparaitre avant que les pucerons ne bifurquent spontanément vers la production de femelles parthénogénétiques (vivipares). L'application cutanée d'hormones juvéniles (JH I, II, et III) aux larves de quatrième stade ou à des adultes producteurs d'ovipares provoque l'apparition prématurée d'intermédiaires et de vivipares dans la descendance. Les différentes formes intermédiaires produites par des applications répétées de J.H. comprenaient des types ailés ou partiellement ailés. Cependant, les vivipares aptères induits par J.H. étaient morphologiquement normaux et beaucoup étaient capables de se reproduire. Des traitements semblables aux J.H. de vivipares élevées en jours longs (LN 16/8) n'ont pas eu d'effets sur le type de la descendance.On ne sait pas si l'action de JH exogène sur l'induction des vivipares est direct ou indirect. La reprogrammation des embryons, autrement destinés à se développer comme ovipares, est examinée en relation avec notre connaissance du contrôle endocrine du polymorphisme des pucerons.

     

Influence of rearing temperature on triacylglycerol storage in the pea aphid,Acyrthosiphon pisum

Total fatty acids in the pea aphid reared at low temperatures increased significantly compared to that at high rearing temperatures. This change is reflected in a large increase of myristic acid, which occurs exclusively in triacylglycerols. When aphids were moved from 25°C to a lower rearing temperature at 10°C, saturated fatty acids accumulated over time, reaching a maximum at 16th day. When aphids were moved to 4°C, a temperature below the developmental threshold, those aphids did not accumulate saturated fatty acids. Similar results were observed when aphids were exposed to sequential decrease in rearing temperature. However, both total fatty acids and myristic acid in the aphids from the treatments of sequential decreasing rearing temperature were significantly higher compared to those in the aphids from the treatments of sudden decreasing rearing temperature. This result, therefore, supports the hypothesis that cold‐adapted aphids can survive under threshold temperature for a longer period of time than noncold‐adapted aphids. Acetyl‐CoA carboxylase activity in the aphids at 25°C was twofold higher than that in the aphids at 10°C, whereas fatty acid synthase activities in the aphids reared at 25 and 10°C are similar. Aphids reared at 10°C showed a threefold reduction in reproduction rates. This reduced production of new nymphs reduces energy demand and would allow for accumulation of energy in the form of triacylglycerols. Therefore, the increased level of saturated fatty acids in aphids reared at low temperature is probably related to lower utilization of fatty acids rather than increased rates of biosynthesis.     

Entomopathogenic fungi stimulate transgenerational wing induction in pea aphids,Acyrthosiphon pisum (Hemiptera: Aphididae)

1. Aphid natural enemies include not only predators and parasitoids but also pathogens, of which fungi are the most studied for biological control. While wing formation in aphids is induced by abiotic conditions, it is also affected by biotic interactions with their arthropod natural enemies. Wing induction via interactions with arthropod natural enemies is mediated by the increase in their physical contact when alarmed (pseudo‐crowding). Pathogenic fungi do not trigger this alarm behaviour in aphids and, therefore, no pseudo‐crowding occurs. 2. We hypothesise that, while pathogenic fungi will stimulate maternally induced wing formation, the mechanism is different and is influenced by pathogen specificity. We tested this hypothesis using two entomopathogenic fungi, Pandora neoaphidis and Beauveria bassiana, an aphid specialist and a generalist respectively, on the pea aphid, Acyrthosiphon pisum Harris. 3. We first demonstrate that pea aphids infected with either pathogen and maintained in groups on broad bean plants produced a higher proportion of winged morphs than uninfected control aphids. We then show that, when maintained in isolation, aphids infected with either pathogen also produced higher proportions of winged offspring than control aphids. There was no difference between P. neoaphidis and B. bassiana in their effects on wing induction in either experiment. 4. Unlike the effect of predators and parasitoids on pea aphid wing induction, the effect of pathogens is independent of physical contact with other aphids, suggesting that physiological cues induce wing formation in infected aphids. It is possible that aphids benefit from wing induction by escaping infected patches whilst pathogens may benefit through dispersion. Possible mechanisms of wing induction are discussed.     

Behavioural correlates of genetic divergence due to host specialization in the pea aphid, Acyrthosiphon pisum

Host specialization plays a central role in the diversification of herbivorous insects and yet we know very little about the evolution of this trait. Populations of the pea aphid, Acyrthosiphon pisum (Harris), are specialized and locally adapted to either alfalfa or clover. Preference for either plant produces assortative mating, and gene flow between alfalfa and clover populations is consistently restricted (Via, 1991a,b, 1994). Behavioural studies of freely-moving aphids on the plant surface for 30 min suggest that host preference is chemically mediated, involves chemoreception, and is most likely due to secondary compounds located in the epidermal or mesophyll cells. Pea aphids do not distinguish between hosts and non-hosts at a distance but determine whether the plant is suitable or not after only a short probe. Thirty-minute recordings of the activities of aphid stylets using an electrical monitoring system (EPG), where aphids are attached to a gold wire with silver paint, provide a different picture, suggesting that EPG experiments do not accurately reflect natural behaviour during the first 30 min of the aphid-plant interaction.     

Plant penetration by pea aphids (Acyrthosiphon pisum) of different plant range

Plant penetration by the stylets of six clones of the pea aphid, Acyrthosiphon pisum, on Vicia faba (acceptable to all clones) and Pisum sativum (acceptable to 3/6 clones) was investigated by the DC electrical penetration graph technique. In a 10 h recording period, 93% of 144 aphids exhibited sustained feeding on phloem sap. Significant interclonal differences were observed for the incidence of potential drops (indicative of brief punctures of plant cells) and the duration of waveform E1 (insect salivation into a sieve element). In addition, the total duration of the sieve element phase and the duration of completed bouts of sustained feeding differed between the two test plants, in a fashion varying between clones. However, these differences could not be related to the acceptability of plants to the different aphid clones. The duration of the stylet pathway phase preceding the first sustained feeding on phloem sap did not vary significantly with either aphid clone or plant. It is concluded that the resistance of P. sativum to certain A. pisum clones does not arise from factors impeding either stylet penetration through the plant tissues or the maintenance of feeding from the sieve elements. It is proposed that host plant affiliation of A. pisum may be mediated primarily by specific olfactory or gustatory cues, before the aphid initiates stylet penetration of the plant.     

Identification and characterisation of antixenosis and antibiosis to pea aphid (Acyrthosiphon pisum) in Pisum spp. germplasm

Acyrthosiphon pisum is a polyphagous aphid of major importance on the pea crop to which few resistant cultivars are available. In this study, we screened a germplasm collection of Pisum spp. under field conditions over two seasons yielding the identification of a number of accessions with intermediate levels of resistance. Selected accessions were further studied under semi‐controlled and controlled conditions in no choice and choice assays to validate the responses, and to further characterise the mechanisms of resistance operative. Results elucidated the valuable resistance of accession P40 (Pisum sativum ssp. sativum) and P665 (P. sativum ssp. syriacum), with the combination of both antixenosis, by reducing aphid preference, and antibiosis, by diminishing aphid proliferation.     

The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE‐LC‐MS/MS and LC‐MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin‐converting enzyme (an M2 metalloprotease), an M1 zinc‐dependant metalloprotease, a glucose‐methanol‐choline (GMC)‐oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium‐binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium‐mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.     

The assimilation and allocation of nutrients by symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum

The failure of a nutritionally balanced diet to ameliorate the impact of symbiont disruption in the pea aphid Acyrthosiphon pisum (Harris) was investigated using two approaches. The assimilation of dietary nutrients by aphids was investigated using chemically-defined diets containing ³ H-labelled inulin and ¹⁴C-labelled sucrose or amino acids. Symbiotic aphids (i.e., aphids containing their bacteria) had a high sucrose demand and assimilated 72% of sucrose ingested in the diet, whereas the assimilation of sucrose by aposymbiotic aphids (in which the bacteria had been disrupted), was significantly reduced to 47%. The assimilation of individual dietary amino acids by symbiotic aphids varied between 61 and 92%, and there was no impact on the feeding or assimilation rate when the aphids were fed a phloem sap-like diet containing a reduced amount of essential amino acids. Consequently, the absolute amount of each essential amino acid assimilated by symbiotic aphids feeding on a phloem sap-like diet was reduced by 36–59%. Aposymbiotic aphids consistently assimilated a lower proportion of ingested amino acids, and lysine in particular was poorly assimilated from the diet. In a second experiment, the allocation of free amino acids in the haemocoel to aphid embryos was investigated following microinjection of ¹⁴C-labelled amino acids. After 2 h, radiolabel could be detected at varying levels from the embryo complement of both symbiotic and aposymbiotic aphids, indicating rapid but selective uptake by the embryos. The essential amino acids phenylalanine and lysine were incorporated into the protein fraction of embryo tissues, but the rate of incorporation per unit biomass was approximately 4-fold higher in the embryos of aposymbiotic aphids, possibly reflecting increased demand due to the lack of amino acid provisioning from the symbiotic bacteria.