Claudio C. Ramírez

Host range expansion of an introduced insect pest through multiple colonizations of specialized clones

Asexuality confers demographic advantages to invasive taxa, but generally limits adaptive potential for colonizing of new habitats. Therefore, pre‐existing adaptations and habitat tolerance are essential in the success of asexual invaders. We investigated these key factors of invasiveness by assessing reproductive modes and host‐plant adaptations in the pea aphid, Acyrthosiphon pisum, a pest recently introduced into Chile. The pea aphid encompasses lineages differing in their reproductive mode, ranging from obligatory cyclical parthenogenesis to fully asexual reproduction. This species also shows variation in host use, with distinct biotypes specialized on different species of legumes as well as more polyphagous populations. In central Chile, microsatellite genotyping of pea aphids sampled on five crops and wild legumes revealed three main clonal genotypes, which showed striking associations with particular host plants rather than sampling locations. Phenotypic analyses confirmed their strong host specialization and demonstrated parthenogenesis as their sole reproductive mode. The genetic relatedness of these clonal genotypes with corresponding host‐specialized populations from the Old World indicated that each clone descended from a particular Eurasian biotype, which involved at least three successful introduction events followed by spread on different crops. This study illustrates that multiple introductions of highly specialized clones, rather than local evolution in resource use and/or selection of generalist genotypes, can explain the demographic success of a strictly asexual invader.

PSEUDOREPLICATION AND ITS FREQUENCY IN OLFACTOMETRIC LABORATORY STUDIES

The evaluation of behavioral responses of an organism to a particular stimulus normally implies the design of a bioassay. Measurements of the response in a number of replicates are necessary to perform inferential statistics and therefore accept or reject a hypothesis about the effect of the stimulus on the behavior of the organism under study. In the present article, we address the importance of pseudoreplication in studies of chemical ecology, particularly in laboratory experiments on olfactory responses of insects to semiochemicals in olfactometers and wind tunnels. Pseudoreplication may be caused by lack of independence in the stimulus or the experimental device, the reutilization of test insects, or the use of groups of test insects, without adequate statistical analysis addressing such dependency. Each and all of the cases reviewed (N = 105) lacked information in at least one of the factors listed above; hence no cases could be said with certainty to be free of pseudoreplication. Forty-nine cases (46.7%) contained explicit information revealing that pseudoreplication existed in terms of one or more of the criteria listed above; in only three of these cases did the authors address statistically the stated dependence of the samples. Pseudoreplication due to different factors ranged from 2% to 30% of the cases, with an average of 13%. The most frequent sources of pseudoreplication were the reuse of the device and the use of groups of test insects. The analysis showed the low importance given to obtaining independent replicates in bioassays involving olfactometric responses of insects to semiochemicals.

Population Genetic Structure of Codling Moth (Lepidoptera: Tortricidae) from Apple Orchards in Central Chile

Abstract Codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the main pest of pome fruits worldwide. Despite its economic importance, little is known about the genetic structure and patterns of dispersal at the local and regional scale, which are important aspects for establishing a control strategy for this pest. An analysis of genetic variability using microsatellites was performed for 11 codling moth populations in the two major apple (Malus domestica Borkh) cropping regions in central Chile. Despite the geographical distances between some populations (≈185 km), there was low genetic differentiation among populations (FST = 0.002176), with only slight isolation by distance. Only ≈0.2% of the genetic variability was found among the populations. Geographically structured genetic variation was independent of apple orchard management (production or abandoned). These results suggest a high genetic exchange of codling moth between orchards, possibly mediated by human activities related to fruit production.

The invasion route for an insect pest species: the tobacco aphid in the New World

Biological invasions are rapid evolutionary events in which populations are usually subject to a founder event during introduction followed by rapid adaptation to the new environment. Molecular tools and Bayesian approaches have shown their utility in exploring different evolutionary scenarios regarding the invasion routes of introduced species. We examined the situation for the tobacco aphid, Myzus persicae nicotianae, a recently introduced aphid species in Chile. Using seven microsatellite loci and approximate Bayesian computation, we studied populations of the tobacco aphid sampled from several American and European countries, identifying the most likely source populations and tracking the route of introduction to Chile. Our population genetic data are consistent with available historical information, pointing to an introduction route of the tobacco aphid from Europe and/or from other putative populations (e.g. Asia) with subsequent introduction through North America to South America. Evidence of multiple introductions to North America from different genetic pools, with successive loss of genetic diversity from Europe towards North America and a strong bottleneck during the southward introduction to South America, was also found. Additionally, we examined the special case of a widespread multilocus genotype that was found in all American countries examined. This case provides further evidence for the existence of highly successful genotypes or ‘superclones’ in asexually reproducing organisms.

Host plant changes produced by the aphid Sipha flava: consequences for aphid feeding behaviour and growth

Induced plant responses may affect the behaviour and growth of the attacking herbivore insect. The aphid Sipha flava (Forbes) produces reddish spots on the infested leaf of its host plant Sorghum halepense (L.). In order to assess the consequences on the aphid of this presumptive induced plant response, we studied the feeding behaviour and growth of S. flava on previously infested and non‐infested leaves of S. halepense. Considering that the reddish pigment could play a defensive role, its effect on aphid survival was determined in artificial diets. In addition, changes in the histology of the leaf and the chemical nature of the induced pigment were also studied. Aphids devoted a significantly shorter total time to non‐penetration activities in infested than in non‐infested leaves. Time before the first phloem ingestion tended to be shorter in infested leaves. The mean relative growth rate of S. flava nymphs was significantly higher on infested than on non‐infested leaves. Survival of aphids on diet containing the reddish extract was not significantly different from that on the control diet. Infestation of S. halepense by S. flava produced a reddish coloration in the leaf, which was identified as an anthocyanin by UV‐visible spectrometry. Light microscopy showed that only mesophyll cells of previously infested plants presented swelled, dispersed, and heterogeneously stained chloroplasts with a higher accumulation of starch granules, no grana arranged in stacks, and reduction in the amount of inner membranes (thylakoids), relatively to chloroplasts of non‐infested leaves. Scanning electron micrographs of leaf surface revealed reduced presence of crystalline epicuticular waxes of epidermal cells in infested leaves as compared to non‐infested ones. The main conclusion is that the attack of S. flava to S. halepense leaves induced plant susceptibility where aphid feeding behaviour and growth were both enhanced on previously infested leaves.

The Influence of Previous Experience and Starvation on Aphid Feeding Behavior

It was hypothesized that (1) previous experience of aphids on a host plant leads to differences in their feeding behavior relative to aphids without previous experience on it and that (2) a change in the physiological state of the aphid modifies their experience-induced behavior. Using electronic recording, the feeding behavior of the aphid Sitobion fragariae (Walker) on wheat Triticum aestivum L. and oat Avena sativa L. was examined, comparing aphids with or without previous experience on a given host and with or without a period of starvation before assessing probing behavior. All comparisons were performed within a single aphid clone to minimize the effect of genetic variation. Feeding behavior on wheat was significantly affected by previous experience and starvation. The effect of previous experience interacted with the host plant where feeding behavior was tested. Aphids feeding on wheat following previous experience on wheat showed a longer time and a higher number of pathway activities and less time in waveform F (i.e., mechanical stylet work and penetration difficulties) than did aphids feeding on wheat after a previous experience on oat. No differences in the time from the beginning of the recording until the first salivation into the sieve elements were found. When aphids were subjected to a period of starvation, the time devoted to xylem ingestion increased compared with that of nonconstrained aphids. These results are discussed in terms of factors affecting foraging decisions.

Response of the Beetle Hylastinus obscurus Marsham (Coleoptera: Scolytidae) to Red Clover (Trifolium pratense L.) Volatiles in a Laboratory Olfactometer

Abstract The response of field-collected Hylastinus obscurus Marsham (Coleoptera: Scolytidae) to volatiles from Trifolium pratense L. of different ages was studied in a four-arm olfactometer. Volatiles from 1.5-, 2-, and 2.5-yr-old plants were more attractive than volatiles from 1-, 3-, and 3.5-yr-old plants. Two-year-old plants were preferred during winter, spring, and summer. One-year-old plants were not preferred in their early stages, but preference increased as they aged. Volatiles from 3-yr-old plants were never preferred. The essential oils obtained from 2-yr-old T. pratense elicited an attraction response from H. obscurus. Beetle response is discussed in relation to host-locating behavior.

Salivation into sieve elements in relation to plant chemistry: the case of the aphid Sitobion fragariae and the wheat, Triticum aestivum

Extended sieve element salivation (El waveform in the electrical penetration graph) is a characteristic activity during early sieve element punctures, particularly in resistant plants. In order to explore a chemically-mediated mechanism of resistance associated with sieve element salivation, we compared the pattern of feeding behaviour of the aphid, Sitobion fragariae (Walker), on two cultivars of the wheat Triticum aestivum L., with different concentrations of hydroxamic acids (Hx). During 24 h of electronic monitoring, aphids dedicated over 50% of the total time to phloem ingestion from the sieve elements. Total time allocated to El in the experiment, time to first El within the experiment, time allocated to El before a sustained phloem ingestion (E2) and the contribution of sieve element salivation to the phloem phase (E1/[E1+E2]) were significantly higher in the high-Hx cultivar. The increased salivation in plants with higher contents of Hx suggests the existence, at least in this system, of a chemically-mediated sieve element constraint.

Landscape composition modulates population genetic structure of Eriosoma lanigerum (Hausmann) on Malus domestica Borkh in central Chile

Landscape genetics have been particularly relevant when assessing the influence of landscape characteristics on the genetic variability and the identification of barriers to gene flow. Linking current practices of area-wide pest management information on pest population genetics and geographical barriers would increase the efficiency of these programs. The woolly apple aphid, Eriosoma lanigerum (Hausmann), an important pest of apple orchards worldwide, was collected on apple trees (Malus domestica Borkh) from different locations in a 400 km north-south transect trough central Chile. In order to determine if there was population structure, diversity and flow were assessed. A total of 215 individuals from these locations were analysed using Inter Simple Sequence Repeat (ISSR) markers. Four ISSR primers generated a total of 114 polymorphic loci. The percentage of molecular variation among locations was 18%. As the algorithm used by structure may be poorly suited for inferring the number of genetic clusters in a data set that has an IBD relationship, the number of genetic clusters in the samples was also analyzed using a Bayesian clustering method implemented in software Baps version 4.14. We inferred the presence of four genetic clusters in the study region. Clustering of individuals followed a pattern explained by some geographical barriers. Using partial Mantel tests, we detected barriers to gene flow other than distance, created by a combination of main rivers and mountains. Although landscape genetics are rarely used in pest management, our results suggest that these tools may be suitable for the design of area-wide pest management programs.

HOST-PLANT CHEMICALS AND DISTRIBUTION OF Neuquenaphis ON Nothofagus

Extractable metabolites from leaves (EM) and volatiles released from six Nothofagus species were analyzed by TLC and GC-MS, respectively. Aphids of the genus Neuquenaphis, closely associated to Nothofagus, were sampled on each Nathofagus species. Cluster analyses of Nothofagus species were performed based on the presence or absence of EM and volatiles. Dissimilarity distances, from the cluster analyses of EM and volatiles, were used to evaluate their association with the aphid distribution. A major component identified from EM and volatiles of three species of Nothofagus, α-agarofuran, was attractive to alates of the oligophagous Neuquenaphis sensoriata, which use them as hosts. These results suggest that chemicals play a significant role in the host-plant associations between Neuquenaphis and Nothofagus.