Effects of bacterial secondary symbionts on host plant use in pea aphids

Aphids possess several facultative bacterial symbionts that have important effects on their hosts'' biology. These have been most closely studied in the pea aphid (Acyrthosiphon pisum), a species that feeds on multiple host plants. Whether secondary symbionts influence host plant utilization is unclear. We report the fitness consequences of introducing different strains of the symbiont Hamiltonella defensa into three aphid clones collected on Lathyrus pratensis that naturally lack symbionts, and of removing symbionts from 20 natural aphid–bacterial associations. Infection decreased fitness on Lathyrus but not on Vicia faba, a plant on which most pea aphids readily feed. This may explain the unusually low prevalence of symbionts in aphids collected on Lathyrus. There was no effect of presence of symbiont on performance of the aphids on the host plants of the clones from which the H. defensa strains were isolated. Removing the symbiont from natural aphid–bacterial associations led to an average approximate 20 per cent reduction in fecundity, both on the natural host plant and on V. faba, suggesting general rather than plant-species-specific effects of the symbiont. Throughout, we find significant genetic variation among aphid clones. The results provide no evidence that secondary symbionts have a major direct role in facilitating aphid utilization of particular host plant species.     

A strain of the bacterial symbiont Regiella insecticola protects aphids against parasitoids

Aphids commonly harbour facultative bacterial endosymbionts and may benefit from their presence through increased resistance to parasitoids. This has been demonstrated for Hamiltonella defensa and Serratia symbiotica, while a third common endosymbiont, Regiella insecticola, did not provide such protection. However, this symbiont was recently detected in a highly resistant clone of the peach-potato aphid, Myzus persicae, from Australia. To test if resistance was indeed conferred by the endosymbiont, we eliminated it from this clone with antibiotics, and we transferred it to two other clones of the same and one clone of a different aphid species (Aphis fabae). Exposing these lines to the parasitoid Aphidius colemani showed clearly that unlike other strains of this bacterium, this specific isolate of R. insecticola provides strong protection against parasitic wasps, suggesting that the ability to protect their host against natural enemies may evolve readily in multiple species of endosymbiotic bacteria.     

Interspecific symbiont transfection confers a novel ecological trait to the recipient insect

In Japan, pea aphids Acyrthosiphon pisum mainly feed on vetch and clover, and many aphid clones produce more progeny on vetch than on clover. In this context, particular genotypes of the facultative symbiont Regiella insecticola enhance reproduction of infected pea aphids specifically on clover, thereby broadening the suitable food plant range of the insect. A species that is sympatric to A. pisum, vetch aphids Megoura crassicauda, are commonly found on vetch but not on clover. Laboratory rearing of M. crassicauda strains revealed active reproduction on vetch but substantially no reproduction on clover. Experimental transfection of Regiella from A. pisum to M. crassicauda by haemolymph injection established stable and heritable infection in the recipients, although no Regiella infection has been detected in natural populations of M. crassicauda. Different strains of Regiella-transfected M. crassicauda grew and reproduced on vetch, but exhibited lower fitness in comparison with corresponding uninfected aphid strains. Strikingly, the Regiella-transfected M. crassicauda exhibited improved survival and some reproduction on clover. These results suggest that Regiella has the potential to confer an ecological trait, adaptation to clover, on novel insect hosts, and also account for why Regiella is able to infect M. crassicauda but is scarcely found in these aphid populations.     

Inducing the sexual forms and hatching the eggs of pea aphids

In temperate climates, pea aphids (Acyrthosiphon pisum) produce a single sexual generation each year in response to declining photoperiod and temperature. Mating occurs in the fall and the eggs have an obligatory winter diapause. Genetic recombination during the sexual phase is thought to be an important source of genetic variability within cyclically parthenogenetic aphid populations. Methods for reliably producing sexual forms and hatching the eggs of aphids are therefore central not only to the study of evolutionary change in aphid populations, but also for a general understanding of the origin of agriculturally important variation in destructiveness within pest species.Here, sexual forms of six pea aphid clones were induced in the laboratory and eggs were successfully hatched by creating conditions that closely mimicked those found in field situations. A declining photoperiod was produced by controlling artificial lighting using a timer with variable cycle length. Using these conditions, sexual forms were successfully produced for all six clones tested, which were then mated in all combinations. Eggs were exposed to a daily cycle of freezing and thawing in an incubator under a short-day photoperiod. Egg hatch averaged 60%, but was as high as 89% for some crosses. These methods will permit testing of evolutionary hypotheses and execution of detailed genetic studies of sources of variability within pea aphid populations. They are thus important tools for both evolutionary and agricultural studies.     

Genetic variation in tissue cultures of red clover

Summary Design II matings were made among randomly selected clones of Arlington red clover (Trifolium pratense L.). Progeny were evaluated in vitro on two regeneration media for callus growth and differentiation. Additive genetic variance was a significant source of variability for nearly all traits evaluated, including somatic embryogenesis. In vitro traits, such as rapid callus growth, colony vascularization, root initiation, chlorophyll production and embryogenesis were highly heritable and should respond to breeding and selection. Dominance genetic variance was significant for only a few in vitro characters. Maternal and cytoplasmic factors were significant primarily in the early subcultures. Highly significant additive genetic correlation of performance on two regeneration media was found. A population selected on one of the regeneration media for such characteristics as improved plantlet regeneration, rapid callus growth, long term colony viability or the frequency of root initiation should show correlated improvement on the other medium. No significant differences for embryogenesis were attributable to differences in the regeneration media used. Furthermore, no interaction of additive genetic effects with regeneration media were observed. These data indicate that improvement in the frequency of plantlet regeneration from callus of red clover could effectively be achieved by breeding and selection for embryogenic types.The research reported in this paper (No. 80-3-152) is in connection with a project of the Kentucky Agric. Exp. Stn. and the paper is published with the approval of the director. Part of a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. degree     

Linking the bacterial community in pea aphids with host-plant use and natural enemy resistance

Abstract.  1. Pea aphids, Acyrthosiphon pisum , harbour a range of facultative accessory bacteria (secondary symbionts), including those informally known as PASS (R-type), PAR, PABS (T-type), and PAUS (U-type).
2. To explore the relationship between possession of these bacteria and ecologically important traits of A. pisum , correlations between the accessory bacteria found in 47 parthenogenetic clones of A. pisum and the host plant on which each clone was collected and its susceptibility to natural enemies were surveyed.
3. The bacterial complement varied with plant of collection. PAUS (U) was present in all of 12 clones affiliated to Trifolium but was otherwise rare, while PABS (T) and PASS (R) occurred at significantly higher frequency in clones from Lotus and Vicia , respectively, than clones from other plants.
4. Possession of PABS (T) was associated strongly with resistance to the parasitoid Aphidius eadyi and weakly with resistance to Aphidius ervi . Aphids carrying PAUS (U) were more resistant to the fungal pathogen Pandora ( Erynia ) neoaphidis , although this correlation was complicated by a strong association with host-plant use.     

Genetic variation in asexual populations of two aphids in the genus Acyrthosiphon, from an Australian lucerne field

Twelve lines of Acyrthosiphon kondoi Shinji and Acyrthosiphon pisum (Harris) were sampled from a small lucerne field in Australia and tested to assess genetic variation by measuring their responses to three lucerne cultivars. Seven or eight life history traits were assessed for each species. Differences in the responses of these traits to the cultivars revealed that three to seven genetically distinct clones were represented in the samples of 12 lines for each species. We conclude that the populations of these two aphids are variable in Australia, although they were recently introduced and reproduce asexually. The responses of the aphids to the three cultivars are described. Because of genetic variation in host plant responses of the aphids, cultivars with potential aphid resistance must be exposed to a large number of clones to confirm the presence of useful aphid resistance.

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Résumé Douze lignées d'Acyrthosiphon kondoi Shinji et d'A. pisum Harris ont été échantillonnées dans un petit champ de luzerne australien et leur variabilité génétique examinée en comparant les réponses à 3 cultivars de luzerne. De 7 à 8 caractères biologiques avaient été retenus pour chaque espèce. Les différences observées entre les réponses suivant la distribution de ces caractères chez les pucerons, ont montré que 3 à 7 clones génétiquement distincts étaient représentés dans les 12 lignées de chaque espèce. Nous en concluons que les populations de ces deux espèces de pucerons sont polymorphes en Australie, quoiqu'elles se multiplient d'une façon asexuée et aient été récemment introduites. Les réactions des pucerons aux 3 cultivars sont indiquées. Par suite de la variabilité génétique des réactions des plantes hôtes aux pucerons, des cultivars à résistance potentielle élevée devraient être exposés à un grand nombre de clones de pucerons pour confirmer la présence d'une résistance utilisable.

     

Examination of the success rate of secondary symbiont manipulation by microinjection methods in the pea aphid system

Insects harbor a wide range of microbial symbionts, but their influence on host phenotypes is described in a limited number of biological models. One experimental approach to gain knowledge on the effects of symbionts to their hosts is to create insect lines with and without symbionts and examine their phenotypes. However, the success rate of symbiont elimination and introduction methods is dependent on several parameters that are scarcely tested or described. The pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), is a model insect of symbiosis studies. It harbors a primary symbiont that supplies the host with essential amino acids, and an array of secondary symbionts whose effects have been assessed by manipulating their presence/absence in the insect. Here, we describe the influence of key parameters on the success rate of symbiont manipulation using the pea aphid–secondary symbiont system. We compared two elimination methods differing in antibiotic treatment using several aphid–symbiont combinations. We also created new aphid host–symbiont combinations by secondary symbiont introduction and examined the effects of larval stage of recipient aphids on introduction success. Our study revealed that the aphid–symbiont combination has strong influence on both symbiont introduction and elimination success rates, and that the type of antibiotics and the larval stage of recipient aphids influence the elimination and introduction success rate, respectively.     

Seasonal variation in the photoperiodic responses of a pea aphid population: evidence for long-distance movements between populations

Summary The purpose of the study was to quantify long distance movements in populations of pea aphid, Acyrthosiphon pisum (Harris), by estimating origins and distances travelled by immigrants into a southern Manitoba population. A strong relationship was demonstrated between latitude of origin and photoperiods at which pea aphid populations are stimulated to produce the diapause stage (Smith 1987). Therefore, the approach was to use photoperiodic response as a physiological marker to identify the source of immigrant aphids. The responses of 89 clones from Glenlea, Manitoba (49°38N), sampled 5 times over 2 seasons, were measured. One sample of clones collected the first season had photoperiodic responses similar to those of a population about 300 km to the south, and significantly different from clones collected in spring of the same year at the same site. Weather analysis corroberates that the migrants were probably carried into Manitoba on a southerly flow of air during the previous 24 to 36 h.     

Genetic variation and covariation of susceptibility to parasitoids in the aphid Myzus persicae: no evidence for trade-offs

Parasitoids are an important mortality factor for insects. Susceptibility to parasitoids should thus be under strong negative selection. Nevertheless, ample genetic variation for susceptibility to parasitoids is commonly observed in natural populations, suggesting that trade-offs may constrain the evolution of reduced susceptibility. This can be studied by assessing genetic variation for susceptibility and its covariation with other components of fitness. In a set of 17 clones of the peach potato aphid, Myzus persicae, for which good estimates of heritable variation for life-history traits were available, we found significant clonal variation for susceptibility to two of their common parasitoids: Aphidius colemani and Diaeretiella rapae. One clone, the only one harbouring a facultative endosymbiotic bacterium, Regiella insecticola, was entirely resistant to both parasitoids. Susceptibilities to the two parasitoids exhibited a positive genetic correlation close to unity, implying a general mechanism of defence. However, the susceptibility to parasitoids was uncorrelated to the clones' fecundity or rate of increase, providing no evidence for costs of the ability to resist parasitoids.     

An experimental validation of orphan genes of Buchnera, a symbiont of aphids

Acadesine, 5-amino-4-imidazolecarboxamide riboside (AICAR), has been claimed to protect the heart, lung, and small intestine against ischemic damage. The biochemical mechanisms of this effect of AICAR are not yet fully understood. To understand the mechanism, we examined the effect of AICAR on glucose starvation, since cellular responses to ischemia could be regarded as a protective response to an insufficient blood supply, cells might display adaptive reactions not only to oxygen deficiency but to nutrient deficiency. AICAR was found to confer strong tolerance to glucose starvation. By using antisense RNA expression vector for alpha subunit of 5'-AMP-activated protein kinase, the effect of AICAR was found to be dependent on 5'-AMP-activated protein kinase containing the alpha2 subunit. The AICAR effect was also dependent on the presence of amino acids, indicating an energy source switch from glucose to amino acids.     

Plant waxy bloom on peas affects infection of pea aphids by Pandora neoaphidis

This study examined the effects of the surface wax bloom of pea plants, Pisum sativum, on infection of pea aphids, Acyrthosiphon pisum, by the fungal pathogen Pandora neoaphidis. In prior field surveys, a higher proportion of P. neoaphidis-killed pea aphids (cadavers) had been observed on a pea line with reduced wax bloom, as compared with a sister line with normal surface wax bloom. Laboratory bioassays were conducted in order to examine the mechanisms. After plants of each line infested with aphids were exposed to similar densities of conidia, the rate of accumulation of cadavers on the reduced wax line was significantly greater than on the normal wax bloom line; at the end of the experiment (13d), the proportion of aphid cadavers on the reduced wax line was approximately four times that on the normal wax bloom line. When plants were exposed to conidia first and then infested with aphids, the rate of accumulation of cadavers was slightly but significantly greater on the reduced wax line, and infection at the end of the experiment (16d) did not differ between the lines. When aphids were exposed first and then released onto the plants, no differences in the proportion of aphid cadavers were observed between the pea lines. Greater infection of pea aphid on reduced wax peas appears to depend upon plants being exposed to inoculum while aphids are settled in typical feeding positions on the plant. Additional experiments demonstrated increased adhesion and germination by P. neoaphidis conidia to leaf surfaces of the reduced wax line as compared with normal wax line, and this could help explain the higher infection rate by P. neoaphidis on the reduced wax line. In bioassays using surface waxes extracted from the two lines, there was no effect of wax source on germination of P. neoaphidis conidia.     

Isolation and physiological characterisation of Thiobacillus thyasiris sp. nov., a novel marine facultative autotroph and the putative symbiont of Thyasira flexuosa

A novel facultatively chemolithoautotropic Thiobacillus, isolated from the gill tissue of the marine bivalve Thyasira flexuosa, is described. It is believed to be the symbiont from this animal, providing the animal with carbon fixed by the Calvin cycle. The organism grows lithoautotrophically on thiosulphate, tetrathionate and elemental sulphur, which are oxidised to sulphate. It oxidizes sulphide, thiosulphate, trithionate, tetrathionate and hexathionate, but not thiocyanate. Kinetic constants for these substrates are presented. In autotrophic batch culture it produces yields that are among the lowest reported for thiosulphate or tetrathionate as energy substrates (1.25 and 2.5 g cell-carbon per mol substrate, respectively). Autotrophic cultures contain ribulose bisphosphate carboxylase and excreted 20% of their fixed carbon into the medium during growth. Mixotrophic growth on acetate and thiosulphate resulted in partial repression of the carboxylase. The organism is slightly halophilic and markedly halotolerant, showing optimum growth at about pH 7.5 and maximum growth rate at 37° C. It contains ubiquinone Q-10 and its DNA contains 52 mol % G+C. These characteristics distinguish it from any other Thiobacillus or Thiomicrospira species previously described. The organism is formally described and named as Thiobacillus thyasiris.     

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.

     

Altered behavior and distribution of pea aphids, Acyrthosiphon pisum (Homoptera: Aphididae), infected with Pandora neoaphidis (Zygomycetes: Entomophthorales)

The distribution and mobility of infected aphid hosts can have a great effect on the ability of a pathogen to spread throughout a population. The distribution of dead and living pea aphids (Acyrthosiphon pisum) infected with Pandora neoaphidis was compared with that of their healthy conspecifics. Infected aphids were significantly more likely to be found on the undersides of alfalfa leaves and off of the plants than were healthy aphids. These two shifts in microhabitat location have potential costs and benefits for both the host and the pathogen.     

Injection of essential amino acids substitutes for bacterial supply in aposymbiotic pea aphids (Acyrthosiphon pisum)

The symbiotic bacteria Buchnera contribute to the nutrition of pea aphids, Acyrthosiphon pisum, through the provision of essential amino acids which are lacking in the diet. However, chemically defined diets, containing nutritionally adequate amounts of essential amino acids, fail to rescue aposymbiotic aphids, in which the bacteria have been disrupted with antibiotics. In this study the injection of a mixture of essential amino acids into the haemocoel of aposymbiotic aphids was shown to alleviate, at least partially, the impact of symbiont loss. Specifically, the total amino acid content in the tissues of aposymbiotic aphids was reduced by approximately 40% to levels comparable with symbiotic insects, and there was a 1.7-fold increase in the number of embryos, suggesting that the availability of essential amino acids promotes aphid protein synthesis by rejuvenating the free amino acid pool of aposymbiotic aphids. In addition, a similar effect on the total amino acid content was observed when phenylalanine alone, but not glutamine, lysine or tryptophan, was injected into the haemocoel of aposymbiotic aphids, and there was also a significant increase in the number of embryos following injection of phenylalanine or tryptophan alone. The impact of amino acid injection on the embryo complement of aposymbiotic aphids was limited to an increase in the number of embryos, with no increase in basal embryo size. It is proposed that older embryos may rely on their own complement of symbiotic bacteria for essential amino acid provisioning. Taken together, the data highlight the importance of bacterial provisioning of essential amino acids, particularly the aromatic amino acids, in the intact symbiosis.     

Interaction of fertilizer regime with host-plant resistance in tomato

The effect of fertilizer regime on trichome- and lamellar-based resistance in the wild tomato species, Lycopersicon hirsutum f. glabratum C.H. Mull accession PI 134417, to three insect pests of tomato, the tobacco hornworm, Manduca sexta (L.), the colorado potato beetle, Leptinotarsa decemlineata (Say), and the tomato fruitworm, Heliocoverpa zea (Boddie), was examined. Increasing the rate at which NPK fertilizer was applied, from 1.8 to 19.6 g/plant/week, reduced the trichome-based resistance of PI 134417 to M. sexta and L. decemlineata by lowering both the density of type VI (sensu Luckwill, 1943) glandular trichomes and the amount of 2-tridecanone contained in the tips of these trichomes. 2-Tridecanone is a toxic methyl-ketone responsible for glandular trichome-mediated resistance in PI 134417 to M. sexta and L. decemlineata. A similar increase in the application rate of NPK fertilizer reduced the lamellarbased resistance of PI 134417 to L. decemlineata and H. zea. The meachanisms for this reduction of resistance are unknown, but may be related to improved nutritional quality of hosts at higher fertilizer regimes.