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1.
The probing and feeding behaviour of the cabbage aphid, Brevicoryne brassicae (L.), (Homoptera, Aphididae) was studied on several plant species that represented various levels of acceptability: Sinapis alba L. (a permanent host plant), Capsella bursa-pastoris (L.) Med., Thlaspi arvense L., Lunaria annua L., Erysimum cheiranthoides L. (accidental host plants), Vicia faba L. (a non-host plant), using the electrical penetration graph technique (EPG). B. brassicae on V. faba did not show any patterns related to penetration of phloem vessels. Stylet penetration was deterred on L. annua and E. cheiranthoides where non-penetration prevailed, the periods of sap ingestion were short or did not occur, the percentage of time spent in the phloem was consistently low (5–6%) and E1 salivation predominated. The pathway activities were not suppressed on C. bursa-pastoris and T. arvense and the aphids spent an average of 3 h in the phloem during the 8-h experiment. However, a considerable delay between finding and accepting the phloem and a substantial proportion of E1 salivation (20–30% of all phloem activities) indicated a deterrent factor in the sieve elements of these plants. Aphid probing and sap ingestion were rarely interrupted on S. alba. The results of this study suggest that the deterrent agents vary in activity and may hinder stylet penetration at different levels (epidermis, parenchymatous tissues and/or phloem elements), depending on the plant species.  相似文献   

2.
Summary Pea aphids show intraclonal variability in antipredator behaviour. Among the offspring of a single parthenogenetically reproducing female, some individuals drop from the plant in response to alarm pheromone while others remain on the plant. We demonstrate that this intraclonal behavioural variability can be altered by selection. The proportion of aphids dropping in response to alarm pheromone was significantly greater in lines in which this behaviour was selected than in clonally identical lines in which the opposite phenotype was favoured. This change occurred within one generation and could not be attributed to grand-maternal effects, nor to environmental effects. These results demonstrate the ability of clonal aphids to adapt to changes in the environment within a single generation.  相似文献   

3.
We studied the development and feeding behaviour of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), on the Radius and Sapko alfalfa ( Medicago sativa L.) (Fabaceae) cultivars. Three saponins and flavones were identified in the alfalfa cultivars after thin layer chromatography separation. Cultivar Radius differed from Sapko in that it had a higher level of saponins, including zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid glycoside. The flavones identified, including 7- O -β-D-glucuronopyranosyl-4'- O- [2'- O- E-feruloyl- O -β-D-glucuronopyranosyl(1→2)- O -β-D-glucuronopyranoside] apigenin, 7- O -{2- O- E-feruloyl-[β-D-glucuronopyranosyl(1→3)]- O -β-D-glucuronopyranosyl(1→2)- O -β-D-glucuronopyranoside} apigenin, and 4'- O- [2'- O -E-feruloyl- O -β-D-glucuronopyranosyl(1→2)- O -β-D-glucuronopyranoside] apigenin, occurred in tissues of both alfalfa cultivars. However, cv. Radius had very low mean flavonoid concentrations in comparison to cv. Sapko. Pea aphids that fed on cv. Radius plants showed a reduction in reproduction and survival. The aphid pre-reproductive period on cv. Radius was prolonged and the reproductive and post-reproductive periods on cv. Radius were reduced, compared to those on cv. Sapko. Cultivar Radius also negatively influenced aphid probing behaviour. This was especially the case during the initial period of the pathway phase. The results suggested that alfalfa cv. Radius, which had a higher level of saponins and a lower level of flavonoids, was less accepted by the pea aphid.  相似文献   

4.
Electrical penetration graphs (DC EPG) were used to monitor the feeding behavior of the pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) exposed to mixtures of saponins: 3GlcA, 28AraRhaXyl medicagenic acid glycoside (MAG) and zanhic acid tridesmoside (ZAD) with flavonoid apigenin, on agarose-sucrose gels. In general, mixtures of saponins with apigenin incorporated into agarose-sucrose gels resulted in a reduction in both the number of aphid probes and delayed their duration. Aphid feeding on gels impregnated with mixtures of the tested chemicals also showed a reduction in salivation into the gels and elongation of passive ingestion from the gels (EPG patterns g-E1 and g-E2, respectively). There were significant differences among the feeding behaviors of the pea aphids on gels containing mixtures of either MAG or ZAD with apigenin. These differences were also depended on the concentration and proportions of these compounds.  相似文献   

5.
Abstract.
  • 1 Temporal changes in host adaptation were followed in a local population of the pea aphid, Acyrthosiphon pisum. Aphid clones were collected in one alfalfa and one clover field at three different times. In the spring, first-generation females were collected. Later, in the autumn, females belonging to the last parthenogenetic generation were collected. Lastly, sexual females were collected after mating in autumn and allowed to produce eggs which were hatched. The performance was evaluated on alfalfa and clover. The spring-collected individuals were also assessed on peas.
  • 2 On the overwintering hosts clover and alfalfa, the clones performed best on the plant of origin, i.e. negative correlations in performance. Correlations between performance on the temporary summer host, pea, and that on clover/alfalfa were weak or nonsignificant.
  • 3 Significant variation in host performance was found within both host fields at spring, which is a prerequisite for changes in clone composition due to selection/migration.
  • 4 The clones from alfalfa showed an increase in mean performance on alfalfa between spring and autumn, whereas no changes among the clones from the clover field were observed. This difference in seasonal response between the two fields could have been the result of larger variation in performance among the alfalfa clones and/or a differential tendency to migrate among clones in both fields.
  • 5 After sexual recombination in the autumn, mean performance in the alfalfa field returned to the spring level, probably as a result of emergence of new genetic combinations. In the clover field, mean performance did not change significantly over time.
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6.
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.  相似文献   

7.
The effects of potato [Solanum tuberosum L. (Solanaceae)] plant damage on the host plant-selection behaviour of the potato aphid, Macrosiphum euphorbiae Thomas (Homoptera: Aphididae), were studied. The damage inflicted to the plant was only of short duration and observations on aphid behaviour were made immediately following plant damage. The underlying questions of the study were to know how much time it takes for plant defence mechanisms to be activated and if this activation had noticeable repercussions on aphid behaviour. We considered stresses of various natures: biotic (pre-infestation by conspecifics or by Colorado potato beetles) and abiotic (scissor cuts). Aphid responses to host plant semiochemicals were investigated using a darkened arena bioassay and the probing behaviour was assessed using the electrical penetration graph technique. Aphids were attracted to their host plant (undamaged or damaged). In a preference test (undamaged plant vs. damaged plant), plants previously infested by conspecifics were preferred to undamaged plants, but this preference was not observed for heterospecific and abiotic damage. However, aphid probing behaviour was not modified on plants previously infested by conspecifics, whereas some changes were observed subsequently to heterospecific and abiotic damages. Our data present evidence that plants can respond to biotic and abiotic stresses soon after the damage is inflicted and when the damage is of short duration. The diverse consequences of these various local plant responses on M. euphorbiae behaviour are discussed in the context of plant defence strategies against aphid colonization.  相似文献   

8.
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.
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.
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9.
  1. Viral insect-borne plant pathogens have devastating impacts in agroecosystems. Vector-borne pathogens are often transmitted by generalist insects that move between non-crop and crop hosts. Insect vectors can have wide diet breadths, but it is often unknown which hosts serve as pathogen reservoirs and which non-crop host harbours the highest density of vectors.
  2. In the Pacific Northwest USA, the pea aphid (Acyrthosiphon pisum) is a key virus vector in pulse crops. Despite pea aphid having a large number of potential non-crop plant hosts occuring in the region, no reservoir has yet been identified for the economically-costly pathogen Pea Enation Mosaic Virus (PEMV).
  3. We addressed these issues by linking field surveys of an aphid vector and plant virus with statistical models to develop risk assessments for common non-crop legumes; in 2018, we completed a 65-site survey where aphids were surveyed in weedy legumes within and outside dry pea fields.
  4. We quantified the abundance of pea aphids on 17 hosts, and plant tissue was tested for PEMV. Relatively high densities of A. pisum were found in habitats dominated by hairy vetch (Vicia villosa), which was the only legume other than cultivated dry pea where PEMV was detected.
  5. Our results indicate that V. villosa is a key alternative host for PEMV, and that pest management practices in this region should consider the distribution and abundance of this weedy host in viral disease mitigation efforts for pulses.
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10.
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.  相似文献   

11.
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.  相似文献   

12.
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.  相似文献   

13.
Using the magnetic particles method, we isolated six polymorphic microsatellite loci from the pea aphid, Acyrthosiphon pisum (Harris), which feeds on a wide range of legume species. The isolated loci were polymorphic, with three to six alleles in 40 aphids. Expected heterozygosities ranged from 0.12 to 0.65. These loci can be used to quantify clonal diversity and compare genetic population structure between sexual and asexual populations.  相似文献   

14.
Two viviparous parthenogenetic clones of the green peach aphid, Myzus persicae (Sulzer), one collected from Rydalmere, New South Wales (NSW), and the other from South Perth (SP), Western Australia, were reared on radish, Raphanus sativus L. cv. Scarlet Globe, under controlled conditions. The NSW clone was fed on simple artificial diets containing alkaloid extracted from narrow-leafed lupin, Lupinus angustifolius L. cv. Fest., and its reproductive performance monitored over 112 h. Forty (40) h into the experiment and thereafter, aphids on the control diet (sucrose solution) produced significantly more offspring (P<0.05) than those on diets containing alkaloid. In a separate experiment, apterae of each clone were caged on three lines (cv. Yorrel, cv. Danja and 84L:441) of narrow-leafed lupin, and allowed to reproduce. The first three offspring were retained, and all developed to 3rd or 4th instar stage. Two nymphs were removed, and the remaining nymph reared through. All three lines produced adults. The number of young produced were counted over 11 days. Fecundity of the SP clone was lower on line 84L:441, but there was no difference in the fecundity of the NSW clone. Phloem exudate and green tissue was concomitantly collected from all lines, and analysed by GC-MS for the alkaloids lupanine and 13-hydroxylupanine. Line 84L:441 contained the highest level of total alkaloids in both phloem and tissue. All experiments indicate that alkaloid level may suppress fecundity of green peach aphids.  相似文献   

15.
Sugars and amino acids were analysed in the phloem sap (sampling by the exudation method) of four clones of lucerne (Medicago sativa L.) characterized by their resistance to the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae). Differences in the qualitative amino acid balance are shown and a negative correlation between the reproductive rate of the aphid and the sugars/amino acids ratio is underlined. To demonstrate any causal relationship between these two variables, the reproduction of A. pisum on artificial diets copying the sap of the two extreme clones (resistant and susceptible) was measured. The results show that the resistance is not a simple nutritional effect; however, the amino acid balance contributes to the resistance exhibited by some cultivars.
Résumé L'analyse (sucres et acides aminés) de la sève de 4 cultivars de luzerne (Medicago sativa) caractérisés pour leur résistance au puceron du pois (Acyrthosiphon pisum (Harris)) (Homoptera: Aphididae), est réalisée sur des exsudats de phloème. La teneur en sucres (exprimée en équivalent saccharose) montre d'importantes différences: rapport de 1 à 3 respectivement entre les clones sensible et résistant. La teneur globale en acides aminés exsudés varie en sens inverse, entraînant une corrélation négative pour les 4 clones étudiés entre le rapport sucre/acides aminés (0,6 à 3,6) et la résistance mesurée par le taux net de reproduction à 14 jours (78,5 à 11,2 larves/femelle). L'analyse des acidses aminés révèle de plus des différences qualitatives entre cultivars, notamment pour certains acides aminés essentiels méthionine, histidine, lysine, aromatiques.Le rôle de l'équilibre en acides aminés et en sacharose dans la résistance de certains cultivars au puceron du pois est apprécié par des mesures de la reproduction de A. pisum sur des milieux artificiels reproduisant le rapport sucre/acides aminés et/ou le spectre des acides aminés des 2 clones extrêmes (sensible et résistant). La résistance n'apparaît pas être le résultat d'une simple différence de la qualité nutritionnelle de la sève; la balance en acides aminés doit cependant participer pour une part à la résistance au puceron.
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16.
Analysis of electrically recorded feeding behaviour of aphids was combined with colony‐development tests to search for sources of resistance to Myzus persicae (Sulzer) (Homoptera: Aphididae) in tuber‐bearing Solanum species (Solanaceae), aiming at a reduction of potato leaf roll virus (PLRV) transmission. Twenty genotypes, originating from 14 gene bank accessions, representing 13 wild tuber‐bearing Solanum spp., three Solanum tuberosum L. (potato) cultivars, and one S. tuberosum breeding line, were selected. Colony‐development tests were carried out in no‐choice experiments by placing adult aphids on plants of each genotype and counting numbers of nymphs and adults on young plants after 8 and 15 days, and on flowering plants after 14 and 30 days. Large differences were observed among genotypes: some developed small colonies and others developed large ones. Also, in a few genotypes, resistance in mature plants was different for leaves of different ages; young leaves were resistant to aphids whereas old senescent leaves were susceptible. The electrical penetration graph (DC‐EPG system) technique was used to study aphid feeding behaviour on each Solanum genotype for 6 h. Electrical penetration graph (EPG) results also showed large differences among the genotypes, indicating resistance at the leaf surface and at three different levels of plant tissue (epidermis, mesophyll, and phloem). Therefore, it was concluded that different mechanisms of resistance to M. persicae exist among the genotypes analysed. EPGs recorded from aphids on Solanum berthaultii Hawkes and Solanum tarijense Hawkes with and without glandular trichomes showed that strong surface resistance can bias EPG parameters associated with resistance located in deeper tissues. Experimental evidence is presented that the resistance to aphids in the genotypes with glandular trichomes strongly depends on these morphological structures.  相似文献   

17.
Aphidius staryi Chen & Luhman n. sp. is described. The species was collected and introduced into California from Israel and Turkey byD. González. The new species is morphologically most similar toAphidius smithi, and keys toEady's urticae group.  相似文献   

18.
This study sheds light on a poorly understood area in insect-plant-microbe interactions,focusing on aphid probing and feeding behavior on plants with varying levels of arbuscular mycorrhizal(AM)fungus root colonization.It investigates a commonly occurring interaction of three species:pea aphid Acyrthosiphon pisum,barrel medic Medicago truncatula,and the AM fungus Rhizophagus irregularis,examining whether aphid-feeding behavior changes when insects feed on plants at different levels of AM fungus colonization(42% and 84% root length colonized).Aphid probing and feeding behavior was monitored throughout 8 h of recording using the electrical penetration graph(EPG)technique,also,foliar nutrient content and plant growth were measured.Summarizing,aphids took longer to reach their 1st sustained phloem ingestion on the 84% AM plants than on the 42% AM plants or on controls.Less aphids showed phloem ingestion on the 84% AM plants relative to the 42% AM plants.Shoots of the 84% AM plants had higher percent carbon(43.7%)relative to controls(40.5%),and the 84% AM plants had reduced percent nitrogen(5.3%)relative to the 42% AM plants(6%).In conclusion,EPG and foliar nutrient data support the hypothesis that modifications in plant anatomy(e.g.,thicker leaves),and poor food quality(reduced nitrogen)in the 84% AM plants contribute to reduced aphid success in locating phloem and ultimately to differences in phloem sap ingestion.This work suggests that M.truncatula plants benefit from AM symbiosis not only because of increased nutrient uptake but also because of reduced susceptibility to aphids.  相似文献   

19.
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.  相似文献   

20.
The effects of two bacterial endosymbionts, designated PASS and PAR, were evaluated on the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera:Aphididae), in which they occur facultatively, and on the blue alfalfa aphid, A. kondoi Shinji, in which these bacteria have not been found in natural populations. Subclones of pea aphids and blue alfalfa aphids, derived from parent aphid clones that did not contain PASS or PAR, were infected with one or both bacteria, generating PASS- and/or PAR-positive subclones with minimal genetic differences from the parent clones. Under laboratory conditions at 20 °C, PAR consistently reduced the fecundity (by between 19 and 60%) of subclones derived from three different parent pea aphid clones on bur clover, Medicago hispida Gaertn. PAR had intermediate effects on pea aphids reared on sweet pea, Lathyrus odoratus L., and had no significant effect on pea aphids on alfalfa, Medicago sativa L. The effect of PASS was either neutral or negative, depending on parent clone as well as host plant. Also at 20 °C, PASS reduced fecundity (70–77%) and longevity (40–48%), and increased the age of first reproduction (by up to 1.5 days) of blue alfalfa aphid reared on alfalfa and clover. PAR had a less dramatic effect (e.g., 30–39% reduction in fecundity) on these traits of blue alfalfa aphid. In contrast, PAR and PASS increased the fitness of pea aphid subclones of one parent clone reared for three generations at 25 °C on each of the three test plants. Without facultative bacteria, fecundity of the parent clone was reduced to a mean total of < 6 offspring per adult at this elevated temperature, but with PASS or PAR, mean total fecundity of its subclones was > 35. However, this ameliorative effect of facultative bacteria at 25 °C was not found for two other sets of parent clones and their derived subclones. Alate production in pea aphids was significantly increased in large populations of two PASS- and PAR-positive subclones relative to their parent clones. Attempts to transmit PASS or PAR horizontally, i.e., from aphid to aphid via feeding on host plants (bur clover), were unsuccessful.  相似文献   

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