Digestive amylase and pectinase activity in the larvae of alfalfa weevil Hypera postica (Coleoptera: Curculionidae)

The alfalfa weevil Hypera postica is a serious economic pest in most alfalfa grown in many countries worldwide. Digestive α-amylase and pectinase activities of larvae were investigated using general substrates. Midgut extracts from larvae showed an optimum activity for α-amylase against starch at acidic pH (pH 5.0). α-Amylase from larval midgut was more stable at mildly acidic pH (pH 5–6) than highly acidic and alkaline pH. The enzyme showed its maximum activity at 35°C. α-Amylase activity was significantly decreased in the presence of Ca²⁺, Mg²⁺ and sodium dodecylsulfate. On the contrary, K⁺ and Na⁺ did not significantly affect the enzyme activity. Zymogram analysis revealed the presence of one band of α-amylase activity in in-gel assays. Pectinase activity was assayed using agarose plate and colorimetric assays. Optimal pH for pectinase activity in the larval midgut was determined to be pH 5.0. Pectinase enzyme is more stable at pH 4.0–7.0 than highly acidic and alkaline pH. However, the enzyme was more stable at slightly acidic pH (pH 6.0) when incubation time increased. Maximum activity for the enzyme incubated at different temperatures was observed to be 40°C. Optimum pH activity for α-amylase and pectinase is not completely consistent with the pH prevailing in the larval midgut. This is the first report of the presence of pectinase activity in H. postica.     

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

Proteolytic activities in alfalfa weevil (Hypera postica) larval midguts have been characterized. Effects of pH, thiol activators, low-molecular weight inhibitors, and proteinase inhibitors (PIs) on general substrate hydrolysis by midgut extracts were determined. Hemoglobinolytic activity was highest in the acidic to mildly acidic pH range, but was maximal at pH 3.5. Addition of thiol-activators dithiothreitol (DTT), 2-mercaptoethanol (2-ME), or L-cysteine had little effect on hemoglobin hydrolysis at pH 3.5, but enhanced azocaseinolytic activity two to three-fold at pH 5.0. The broad cysteine PI E-64 reduced azocaseinolytic activity by 64% or 42% at pH 5 in the presence or absence of 5 mM L-cysteine, respectively. Inhibition by diazomethyl ketones, Z-Phe-Phe-CHN(2) and Z-Phe-Ala-CHN(2), suggest that cathepsins L and B are present and comprise approximately 70% and 30% of the cysteine proteolytic activity, respectively. An aspartyl proteinase component was identified using pepstatin A, which inhibited 32% (pH 3.5, hemoglobin) and 50% (pH 5, azocasein) of total proteolytic activity. This activity was completely inhibited by an aspartyl proteinase inhibitor from potato (API), and is consistent with the action of a cathepsin D-like enzyme. Hence, genes encoding PIs with specificity toward cathepsins L, B and D could potentially be effective for control of alfalfa weevil using transgenic plants.     

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'.     

Effects of pea aphid secondary endosymbionts on aphid resistance and development of the aphid parasitoid Aphidius ervi: a correlative study

In order to reduce parasite‐induced mortality, hosts may be involved in mutualistic interactions in which the partner contributes to resistance against the parasite. The pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), harbours secondary bacterial endosymbionts, some of which have been reported to confer resistance against aphid parasitoids. Although this resistance often results in death of the developing parasitoid larvae, some parasitoid individuals succeed in developing into adults. Whether these individuals suffer from fitness reduction compared to parasitoids developing in pea aphid clones without symbionts has not been tested so far. Using 30 pea aphid clones that differed in their endosymbiont complement, we studied the effects of these endosymbionts on aphid resistance against the parasitoid Aphidius ervi Haliday (Hymenoptera: Braconidae: Aphidiinae), host–parasitoid physiological interactions, and fitness of emerging adult parasitoids. The number of symbiont species in an aphid clone was positively correlated with a number of resistance measurements but there were also clear symbiont‐specific effects on the host–parasitoid interaction. As in previous studies, pea aphid clones infected with Hamiltonella defensa Moran et al. showed resistance against the parasitoid. In addition, pea aphid clones infected with Regiella insecticola Moran et al. and co‐infections of H. defensa–Spiroplasma, R. insecticola–Spiroplasma, and R. insecticola–H. defensa showed reduced levels of parasitism and mummification. Parasitoids emerging from symbiont‐infected aphid clones often had a longer developmental time and reduced mass. The number of teratocytes was generally lower when parasitoids oviposited in aphid clones with a symbiont complement. Interestingly, unparasitized aphids infected with Serratia symbiotica Moran et al. and R. insecticola had a higher fecundity than unparasitized aphids of uninfected pea aphid clones. We conclude that in addition to conferring resistance, pea aphid symbionts also negatively affect parasitoids that successfully hatch from aphid mummies. Because of the link between aphid resistance and the number of teratocytes, the mechanism underlying resistance by symbiont infection may involve interference with teratocyte development.     

Host-dependent association and its implications on the relative abundance of blue alfalfa aphid and pea aphid (Homoptera: Aphididae) on alfalfa in Oklahoma

Field populations of blue alfalfa aphid and pea aphid on alfalfa were sampled during 1985 and 1986 to determine the association of co-occurrence, interspecific interactions and comparative temporal variations in the spatial dispersion patterns of these species in Oklahoma. Relative abundance of these species is discussed in the light of above analyses.Cole' s coefficient revealed a high degree of association between these species in terms of their occurrence on the same alfalfa stems in the field. Regression analyses indicated that the species populations tended to increase in concert on the same stems without evidence of competitive displacement. Spatial dispersion patterns of both species were highly aggregated at low population densities early in the season. Over time, both species tended to disperse and became less aggregated as numbers increased. It was concluded that magnitude of interspecific interactions between the blue alfalfa aphid and the pea aphid were not of a nature that they could be termed as competing species. On the contrary, a concept of an “ecospecies” is proposed for practical applications such as sampling plans and economic threshold determinations.     

Nutrient regulation in the pea aphid Acyrthosiphon pisum: application of a novel geometric framework to sugar and amino acid consumption

Abstract. A recently developed framework was applied to investigate the responses of newborn pea aphids, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), to simultaneous variations in dietary suerose and amino acid levels. The location of functional 'targets' for intake and growth were determined experimentally using performance criteria. Behavioural rules employed by insects to approach these targets were investigated by reference to the geometry of arrays of intake and growth across a range of diets. First-instar aphids were fed one of thirty-one chemically-defined diets ranging in sucrose concentration from 200 to 1000 mM and amino acid concentration from 25 to 250 mM. Insect survivorship, dry weight, protein- and carbohydrate-derived growth were high for all diets except those with the lowest nutrient levels. Peaks in final dry weight and protein content identified the intake target as the point reached by larvae fed the 600 mM sucrose, 75 mM amino acid diet. Aphids regulated sucrose primarily by consumption (i.e. behaviourally), whereas amino acids were regulated post-ingestively (i.e. by physiological mechanisms).     

Performance of pea aphid (Acyrthosiphon pisum) clones on host plants and synthetic diets mimicking the same plants phloem amino acid composition

The performances of three clones of pea aphids, with different host affiliations, were evaluated on four host plants species and on four artificial diets. The amino acid compositions of the diets mimicked those of the phloem sap of the respective host plants. The total concentration of amino acids was the same in all the diets. The pea aphid clones performance were significantly affected by amino acid composition of the diets in different ways, implying physiological and/or behavioural differences among coexisting pea aphid clones in response to amino acids in artificial diets. The observed differences in performance on diets between clones were not related to host plant affiliations. Thus, even if the variation in amino acid composition in phloem sap among the host plants affects the pea aphid clones when tested on artificial diets, this variation has no observable effect on pea aphid performance on natural host plants.     

Effect of low and high-saponin lines of alfalfa on pea aphid

Pea aphid fed on a high-saponin line of alfalfa showed reduction of reproduction and survival, and disturbance in development of its population. This line negatively influenced aphid probing behaviour, particularly prolonging the non-probing period and probing of the peripheral tissues (epidermis and mesophyll) and shortening the period of phloem sap ingestion. The high-saponin line of alfalfa differed from the low-saponin one by the presence of zanhic acid tridesmoside and a higher level of 3-GlcA,28-AraRhaXyl medicagenic acid glycoside. The saponins incorporated into sucrose-agarose gels significantly reduced number of the aphid probes into the gels and extended their duration in comparison to the control gels (without tested compounds). Role of zanhic acid tridesmoside and 3-GlcA,28-AraRhaXyl medicagenic acid glycoside as potential factors for partial resistance of alfalfa towards the pea aphid is discussed.     

Differential photoperiodic responses in genetically identical winged and wingless pea aphids, Acyrthosiphon pisum, and the effect of day length on wing development

Abstract. Winged and wingless individuals of a pink clone of the pea aphid, Acyrthosiphon pisum (Harris), showed differences in the response curves for photoperiodic induction of both males and sexual females (oviparae). The critical night length (CNL) for ovipara induction in winged aphids was 0.75 h shorter than in wingless aphids, whereas the CNL for male induction in winged aphids was 1.0h longer than in wingless aphids. This means that in winged aphids the CNL for male induction in winged aphids was 0.5 h longer than that for ovipara induction, while in wingless aphids the CNL for male induction was 1.0–1.5 h shorter than that for ovipara induction, and also the shapes of the curves differed.
Winged aphids were produced by wingless mothers which were crowded as young adults. However, when young adults were crowded in long nights, winged aphids were not produced, and the CNL for wing inhibition was between 9.5 and 10h. This effect of photoperiod on wing induction was maternal.     

Short-term effects of aphid feeding on photosynthetic CO2 exchange and dark respiration in legume leaves

Net CO₂ exchange rates and dark respiration rates were determined for single attached legume leaves (leaflets) after 6 to 9 days of aphid infestation. Plant-aphid combinations used were broad bean ( Vicia faba L. cv. Aquadulce) and cowpea [ Vigna unguiculata (L.) Walp. cv. Caloona)] infested with cowpea aphids ( Aphis craccivora Koch) and broad bean and garden pea ( Pisum sativum L. cv. Victory Freezer) infested with pea aphids [ Acyrthosiphon pisum (Harris)]. Leaves from all aphid-infested plants had significantly greater net CO₂ exchange rates in the light than their respective controls and rates of dark respiration of leaves from infested cowpea and garden pea were also significantly greater than those of controls. Dark respiration, as a percentage of net CO₂ exchange rates in the light, was greater in aphid-infested than in control plants. When the mean net daily carbon gain was calculated for the leaves of each plant-aphid combination, leaves from aphid-infested plants had the greatest gain. It is proposed that net CO₂, exchange rates increased due to increased sink demand and dark respiration rates increased to meet the increased energy requirements of phloem loading and cellular maintenance associated with aphid feeding. The apparent compensatory carbon gain of infested leaves was consumed by the aphids.     

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.     

Fitness effects of two facultative endosymbiotic bacteria on the pea aphid,Acyrthosiphon pisum,and the blue alfalfa aphid,A. kondoi

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.     

Role of phloem sap quality and exudation characteristics on performance of pea aphid grown on lucerne genotypes

The phloem sap of two lucerne (Medicago sativa L.) genotypes inducing differences in pea aphid,Acyrthosiphon pisum Harris (Homoptera: Aphididae) performances (weight, fecundity, survival), was collected by stylectomy (radio frequency microcautery). Sugars and amino acids were assayed. No significant difference in their concentrations could be established between genotypes. It is thus unlikely that a substantial part of the varietal resistance of these lucerne genotypes to pea aphid could be related to the content in sugars or amino acids. The same conclusion was drawn for the sugar/amino acid ratio. The amino acid profiles were globally similar. They nevertheless allowed a discrimination between the two genotypes on a few amino acids (alanine, leucine, isoleucine, arginine, ornithine) only. Moreover, the degree of resistance can be related neither to GABA concentration, nor to that of sulfur amino acids, because these constituants were either absent, or in trace amounts, whatever the lucerne genotype taken into account. Stylectomy parameters revealed some interesting differences in the sap exudation of the two genotypes. Average volume of exudation by stylet and average duration of exudation were respective 2.8 and 2.3 times higher in the susceptible genotype. These results suggest a reduced flow of phloem sap, after puncturing of the sieve-tubes by the aphid stylets on the resistant genotype; this could result in a greater difficulty in phloem sap ingestion by aphids settled on the resistant genotype and thus in suboptimal performances.     

Distribution in time and space of resistance to the pathogenic fungus Erynia neoaphidis in the pea aphid Acyrthosiphon pisum

Two biotypes of pea aphid, Acyrthosiphon pisum, have been recognized in Australia, by their susceptibility (S) or resistance (R) to certain isolates of the fungal pathogen Erynia neoaphidis. The responses of these two biotypes to 40 isolates of the fungus have shown that the R form is largely confined to two States, New South Wales and Victoria, but appears to have recently spread into Queensland and Tasmania. There is no evidence to suggest it occurs outside Australia. Sequential sampling of two field populations of pea aphids during 1981 and 1982 showed that the proportion of R form remained stable at 10.7±3.0 and 14.6±2.6% (mean±standard error) for the two populations. Glasshouse competition experiments run at the comparatively high temperature of 25°C resulted in the R form becoming dominant even when the initial ratio was 4:1 in favour of the S form. The ecological and genetical implications are discussed.

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Résumé Deux types biologiques d'Acyrthosiphon pisum ont été définis, en Australie, suivant leur sensibilité (S) ou leur résistance (R) à certains isolats du champignon pathogène Erynia neoaphidis. Les réponses des deux biotypes à 40 isolats du champignon ont montré que la forme R est essentiellement cantonnée à deux états: Nouvelle Galle due Sud et Victoria, mais a récemment gagné le Queensland et la Tasmanie. Aucun élément ne fait dire qu'elle existe hors d'Australie. Des échantillonnages séquentiels des deux populations de pucerons dans la nature en 1981 et 1982 ont montré que la proportion de la forme R est restée stable à 10,7 et 14,62 dans les deux populations. Des expériences de compétition en serre à la température relativement haute de 25°C ont rendu la forme R dominante, même quand le rapport initial était de 4/1 en faveur de la forme S. La discussion porte sur les conséquences écologiques et génétiques.

     

Effects of parasitism by Praon pequodorum on age-specific fecundity and population growth of the pea aphid, Acyrthosiphon pisum

The effects of parasitism by Praon pequodorum Viereck (Hymenoptera: Aphidiidae) on the survival, development and reproduction of apterous pea aphids, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), were studied in the laboratory. Ten cohorts of aphids differing in age from 0.5 h to 9 days were tested. At 20°C, pea aphids survived an average of 7 days after parasitization. Parasitized first and second instars died before reaching the adult stage. Third instars died as adults but without producing any offspring. Parasitized fourth instars and adult aphids produced a variable number of offspring; their mean total fecundity increased as an exponential function of the age at parasitization. Host age did not significantly affect the period from the beginning of parasitism to the end of reproduction. Equations are provided to estimate the length of the reproductive period, the total fecundity, the intrinsic rate of increase, and the doubling time as functions of aphid age at parasitization. The data are discussed with regard to host population growth. It is shown that a parasite's potential impact on an aphid population is critically dependent on its preference for and successful oviposition in early host instars.

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Résumé Les effets du parasitisme par P. pequodorum Viereck (Hym.: Aphidiidae), sur la survie, le développement et la reproduction des aptères de A. pisum, ont été examinés au laboratoire. L'étude a porté sur 10 cohortes de pucerons dont les âges différaient de 0,5 heure à 9 jours. A 20°C, les pucerons parasités ont survécu en moyenne pendant 7 jours après avoir été parasités. Leur évolution ultérieure dépend au stade auquel ils ont été parasités: au premier et second stades, ils sont morts avant de devenir adultes; au troisième stade, ils sont morts adultes avant de se reproduire; au quatrième stade et adultes, ils ont donné un nombre variable de descendants. La fécondité totale moyenne a crû comme une fonction exponentielle de l'âge au moment où ils avaient été parasités. L'âge de l'hôte n'a pas modifié significativement la durée de la période entre le moment où ils ont été parasités et la fin de la phase de reproduction. Des équations ont été établies en fonction de l'âge des pucerons au moment où ils ont été parasités, pour estimer la longueur de la période de reproduction, la fécondité totale, le taux intrinsèque d'accroissement et le temps de doublement des cohortes de pucerons. Les résultats sont discutés sous l'angle de la dynamique de la population. On en déduit que l'impact potentiel d'un parasite sur une population de pucerons dépend d'abord de ses préférences pour les premiers stades de l'hôte et du succès de sa ponte sur ces stades.

     

The attachment and stylostome of Trombidium newelli (Acari: Trombidiidae), an ectoparasitic mite on adults of alfalfa weevil, Hypera postica (Coleoptera: Curculionidae)

Larvae of the mite Trombidium newelli Welbourn and Flessel are ectoparasitic on adult alfalfa weevil, Hypera postica (Gyllenhall), an insect pest of alfalfa. The mite larvae are found under the elytra, attached to the dorsal surface of weevils abdomen. T. newelli larvae use their chelicerae to penetrate the pliable and weakly sclerotized areas of the hosts integument, and to hold on firmly to the host. The attachment sites associated with larval mites of different degrees of engorgement were examined using both light and scanning electron microscopy. The ventral inside surface of parasitized host tergites revealed a characteristic injury as spots varying in color from white-yellow to golden-brown depending on the engorged size of the mite. In addition, spots associated with fully engorged mite larvae showed an aggressive dendritically radiating mass expanding outward from the site of cheliceral penetration. This mass, known as feeding tube or stylostome was branched, with each branch ending in a cluster of closed bulbs. Stylostomes appear to exist independently in the hosts tissue. Stylostome mass corresponded with the engorgement state of the mite, increasing as the mite larva increased in size. The possible nature and mechanism of stylostome formation is discussed.     

Effects of host age on embryogenesis and encapsulation of the parasite Bathyplectes curculionis in the alfalfa weevil

First- through fourth-instar larvae of Hypera postica were fixed at 12- to 24-hr intervals after parasitization by Bathyplectes curculionis and sectioned histologically. Sections revealed that host age had little effect on time required for embryogenesis and hatching of parasites. B. curculionis completed larval development and emerged from all host instars ca. 11 days after parasitization at 22.2°C. Parasite eggs were fully encapsulated in just 3.3% of the first-instar H. postica compared with 50.0% in the third instar. Much larger hemocyte capsules were formed in later instars as well.     

Effects of food and temperature on development,fecundity and life‐table parameters of Adalia bipunctata (Coleoptera: Coccinellidae)

Development, reproduction and life tables of Adalia bipunctata (L.) were studied at three temperatures (19, 23 and 27°C) on a mixture of frozen pollen and Ephestia kuehniella Zeller eggs as a factitious food and on the aphids Myzus persicae (Sulzer) and Acyrthosiphon pisum (Harris) as natural foods. Development time of A. bipunctata on all tested diets decreased with increasing temperature. Mortality was lowest at 23°C, averaging 44.5%, 42.6% and 24.3% on factitious food, A. pisum and M. persicae respectively. The shortest developmental time from egg to adult at this temperature was observed on factitious food (18.55 days). However, the factitious food was inferior to the aphid diets in terms of reproduction, yielding the longest pre‐oviposition period, shortest oviposition period and lowest fecundity. The mean oviposition rate at 23°C varied from 19.94 to 25.03 eggs day^?1 on factitious food and M. persicae respectively. The intrinsic rate of increase (r_m) on different foods increased with increasing temperature and ranged from a minimum of 0.08 females/female/day on factitious food (19°C) to a maximum of 0.18 females/female/day on A. pisum (27°C). The results suggest that a mixture of E. kuehniella eggs and pollen fully support development of A. bipunctata larvae and can be used as an alternative to live aphids in the mass rearing of the pre‐imaginal stages of the predator. However, reproductive performance of a laboratory population may be better on aphids than on the factitious food.