Estimating SIT-driven population reduction in the Mediterranean fruit fly, Ceratitis capitata, from sterile mating.

Area-wide sterile insect technique (SIT) programs assume that offspring reduction of the target population correlates with the mating success of the sterile males released. However, there is a lack of monitoring tools to prove the success of these programs in real-time. Field-cage tests were conducted under the environmental conditions of the Mediterranean coast of Spain to estimate: (a) the mating success of sterile Vienna-8 (V8) Ceratitis capitata males using molecular markers and (b) their efficacy to reduce C. capitata populations under six release ratios of wild females to wild males to V8 males (1:0:0, 1:1:0, 1:1:1, 1:1:5, 1:1:10, and 1:1:20). Statistical models were developed to predict: (a) the number of females captured in traps, (b) sperm ID (sterile or not) in spermathecae of the trapped females, and (c) the viable offspring produced, using release ratio and temperature as predictors. The number of females captured was affected by relative humidity. However, its influence in the model was low. Female captures were significantly higher in ratios 1:0:0 compared to ratios where V8 males were released. The proportion of V8 sperm in spermathecae increased with temperature and with the number of V8 males released, but leveled off between ratios 1:1:10 and 1:1:20. In all seasons, except winter (no offspring), viable offspring increased with temperature and was lowest for ratio 1:1:20. For the first time, a strong negative relationship between proportion of V8 sperm detected by molecular tools and C. capitata offspring was established. The models obtained should contribute to enhance the efficacy of SIT programs against this pest.

Genetic structure of a phytophagous mite species affected by crop practices: the case of Tetranychus urticae in clementine mandarins.

Tetranychus urticae Koch is a cosmopolitan mite considered as the most polyphagous species among spider mites. This mite is a key pest of clementine mandarins in Eastern Spain, where Spanish clementine production concentrates. Crop management practices can affect the population dynamics of this mite and, consequently, its impact on the orchard. Microsatellite markers were used to study mite population genetics from two commercial orchards which had been managed differently following Integrated Pest Management (IPM) or Organic Pest Management (OPM) schemes during four consecutive years. A multiplex system including 20 microsatellite loci was designed specifically and allowed an efficient and inexpensive genotyping of individual mites. We found that the IPM population had a stronger fluctuation of population structure and higher genetic diversity compared to OPM population. Thus, our study concludes that crop management has an impact on the population genetics of T. urticae which may be related to the alternation of some acaricides under IPM.

Improving the sterile sperm identification method for its implementation in the Area-wide Sterile Insect Technique Program against Ceratitis capitata (Diptera: Tephritidae) in Spain.

The success of sterile males in area-wide sterile insect technique (aw-SIT) programs against Ceratitis capitata (Wiedemann) is currently measured by using indirect methods as the wild:sterile male ratio captured in monitoring traps. In the past decade, molecular techniques have been used to improve these methods. The development of a polymerase chain reaction-restriction fragment-length polymorphism-based method to identify the transfer of sterile sperm to wild females, the target of SIT, was considered a significant step in this direction. This method relies on identification of sperm by detecting the presence of Y chromosomes in spermathecae DNA extract complemented by the identification of the genetic origin of this sperm: Vienna-8 males or wild haplotype. However, the application of this protocol to aw-SIT programs is limited by handling time and personnel cost. The objective of this work was to obtain a high-throughput protocol to facilitate the routine measurement in a pest population of sterile sperm presence in wild females. The polymerase chain reaction-restriction fragment-length polymorphism markers previously developed were validated in Mediterranean fruit fly samples collected from various locations worldwide. A laboratory protocol previously published was modified to allow for the analysis of more samples at the same time. Preservation methods and preservation times commonly used for Mediterranean fruit fly female samples were assessed for their influence on the correct molecular detection of sterile sperm. This high-throughput methodology, as well as the results of sample management presented here, provide a robust, efficient, fast, and economical sterile sperm identification method ready to be used in all Mediterranean fruit fly SIT programs.

Isolation and characterization of polymorphic microsatellite markers in Tetranychus urticae and cross amplification in other Tetranychidae and Phytoseiidae species of economic importance.

Tetranychus urticae Koch is a cosmopolitan phytophagous mite considered as the most polyphagous species among spider mites. Population genetic studies using molecular markers such as microsatellites have proven to be extremely informative to address questions about population structure, phylogeography and host preferences. The aim of this study was to increase the available molecular tools to gain insight into the genetic structure of T. urticae populations of citrus orchards, which might help in their management. Five microsatellite DNA libraries were developed using probes with the motifs CT, CTT, GT and CAC following the FIASCO protocol. Positive clones, those that included the insert with the microsatellite, were detected using the PIMA-PCR technique. Combinations of primers were designed on 22 out of 32 new microsatellites loci and their polymorphism was tested in four populations sampled along the eastern coast of Spain. Eleven successful amplifications were obtained. Cross amplification was tested in the tetranychids Aphlonobia histricina, Eutetranychus banksi, E. orientalis, Oligonychus perseae, Panonychus citri, Tetranychus evansi, T. okinawanus and T. turkestani, and the phytoseiids Amblyseius swirskii, A. cucumeris, A. andersoni, Euseius stipulatus, Neoseiulus barkeri, N. californicus, Phytoseiulus persimilis and Typhlodromus phialatus. Eight successful cross amplifications were obtained.

Superparasitism in laboratory rearing of Spalangia cameroni (Hymenoptera: Pteromalidae), a parasitoid of medfly (Diptera: Tephritidae).

The frequency of superparasitism and its effects on the quality of laboratory-reared Spalangia cameroni (Hymenoptera: Pteromalidae) parasitoids were investigated under laboratory conditions. Numerous variables were measured, such as the number of 'ovip holes' per host as a measure of superparasitism. Adult emergence and sex ratio, as well as female size, emergence ability from soil and longevity were also measured. Finally, an assessment was made of fertility and survival of adult parasitoids emerging from the medfly Ceratitis capitata (Diptera: Tephritidae) pupae with different levels of superparasitism. A high frequency and prevalence of superparasitism under laboratory rearing conditions was observed. The number of 'ovip holes' per host ranged from one to 17, with an average (±SD) of 2.8±3.4. Sex ratios became increasingly female-biased with increasing levels of superparasitism, although overall levels of wasp emergence (male, female) declined. Nevertheless, no relationship was discerned between female size and level of superparasitism. The 'emergence ability from the soil' was higher in those parasitoids that emerged from strongly superparasitized hosts, but not related to the type of substrate in which the host pupae were buried. The level of superparasitism did not have a significant effect on the longevity, fertility and survival of female parasitoids. Our results support the hypothesis that superparasitism in S. cameroni might be adaptive, since attributes such as 'emergence ability from the soil', longevity, fertility and survival were not affected by the level of superparasitism or the presumably detrimental effects derived from physical combats among conspecific larvae. Our findings are relevant to recommendations for rearing S. cameroni for biological control releases, as well as shedding light on superparasitism under both laboratory and field conditions.

Tracking medfly predation by the wolf spider, Pardosa cribata Simon, in citrus orchards using PCR-based gut-content analysis.

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), which is often controlled chemically, is a major citrus pest in Spain; however, alternative biological control strategies such as those based on the conservation of polyphagous predators should be developed. The wolf spider, Pardosa cribata Simon, is an abundant predator found in citrus orchards in eastern Spain. In this study, we have evaluated polymerase chain reaction (PCR)-based techniques as a means of detecting C. capitata DNA remains in P. cribata specimens. To do so, two pairs of C. capitata species-specific primers were designed and tested. Primer specificity was tested on species closely related to C. capitata and with other pests and natural enemies present in citrus orchards. Medfly DNA was detectable in 100% of P. cribata from 0 to 12 h post ingestion for both primer pairs, decreasing to 37% at 96 h after prey ingestion for one pair of primers. DNA detectability half-lives were of 78.25 h and 78.08 h for each pair of primers but no statistical differences were found between them. Pardosa cribata specimens were field-collected daily after sterile C. capitata pupae had been deployed in the citrus orchard. Afterwards, the wolf spiders were analyzed and DNA remains of C. capitata were detected in 5% of them, with a peak of 15% coinciding with maximum C. capitata emergence. This study is the first to reveal the potential use of DNA markers to track medfly predation by P. cribata in citrus orchards and provides a new tool to estimate the potential role of this spider in biological-control conservation programs.

A novel molecular approach to assess mating success of sterile Ceratitis capitata (Diptera: Tephritidae) males in sterile insect technique programs.

Areawide sterile insect technique (SIT) programs against Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), are increasingly implemented worldwide. A key issue in SIT is to assess mating success of released sterile males, which could be currently estimated by egg hatchability and by stored sperm head measurements. We report here on a novel molecular approach that would allow detecting the presence of Mediterranean fruit fly sterile male sperm in the female spermathecae under field conditions, as a precise marker to assess mating performance. The simplicity (only two polymerase chain reactions) and reliability of this method, jointly with the capability to detect Vienna sperm in wild Mediterranean fruit fly maintained in monitoring traps for 7 d under field conditions, suggest that it could be an efficient tool when coupled with areawide SIT programs.

Seasonal photoperiodism regulates the expression of cuticular and signalling protein genes in the pea aphid.

Seasonal photoperiodism in aphids is responsible for the spectacular switch from asexual to sexual reproduction. However, little is known on the molecular and physiological mechanisms involved in reproductive mode shift through the action of day length. Earlier works showed that aphid head, but not eyes, directly perceives the photoperiodic signal through the cuticle. In order to identify genes regulating the photoperiodic response, a 3321 cDNA microarray developed for the pea aphid, Acyrthosiphon pisum was used to compare RNA populations extracted from heads of short- and long-day reared aphids. Microarray analyses revealed that 59 different transcripts were significantly regulated, among which a majority encoded cuticular proteins and several encoded proteins involved in cellular signalling or signal transduction. These results were confirmed by quantitative RT-PCR experiments on two cuticular and three signalling protein genes. Complementary experiments eliminated moulting and circadian rhythms as putative confounding effects. Quantitative RT-PCR performed at additional developmental stages demonstrated the regulation of expression of cuticular and signalling protein genes during the whole process of photoperiod shortening. This suggests that photoperiodic changes could affect cuticle structure and cell to cell communication in the head of aphids in relation with the switch of reproductive modes.

Annotated expressed sequence tags for studies of the regulation of reproductive modes in aphids.

The damaging effect of aphids to crops is largely determined by the spectacular rate of increase of populational expansion due to their parthenogenetic generations. Despite this, the molecular processes triggering the transition between the parthenogenetic and sexual phases between their annual life cycle have received little attention. Here, we describe a collection of genes from the cereal aphid Rhopalosiphum padi expressed during the switch from parthenogenetic to sexual reproduction. After cDNA cloning and sequencing, 726 expressed sequence tags (EST) were annotated. The R. padi EST collection contained a substantial number (139) of bacterial endosymbiont sequences. The majority of R. padi cDNAs encoded either unknown proteins (56%) or housekeeping polypeptides (38%). The large proportion of sequences without similarities in the databases is related to both their small size and their high GC content, corresponding probably to the presence of 5'-unstranslated regions. Fifteen genes involved in developmental and differentiation events were identified by similarity to known genes. Some of these may be useful candidates for markers of the early steps of sexual differentiation.

Side-stepping secondary symbionts: widespread horizontal transfer across and beyond the Aphidoidea.

To elucidate the co-evolutionary relationships between phloem-feeding insects and their secondary, or facultative, bacterial symbionts, we explore the distributions of three such microbes--provisionally named the R-type (or PASS, or S-sym), T-type (or PABS), and U-type--across a number of aphid and psyllid hosts through the use of diagnostic molecular screening techniques and DNA sequencing. Although typically maternally transmitted, phylogenetic and pairwise divergence analyses reveal that these bacteria have been independently acquired by a variety of unrelated insect hosts, indicating that horizontal transfer has helped to shape their distributions. Based on the high genetic similarity between symbionts in different hosts, we argue that transfer events have occurred recently on an evolutionary timescale. In several instances, however, closely related symbionts associate with related hosts, suggesting that horizontal transfer between distant relatives may be rarer than transmission between close relatives. Our findings on the prevalence of these symbionts within many aphid taxa, along with published observations concerning their effects on host fitness, imply a significant role of facultative symbiosis in aphid ecology and evolution.