Planococcus ficus (Hemiptera: Pseudococcidae) movement and demography: methods for generating cohorts for laboratory studies.

Planococcus ficus (Signoret) is a worldwide pest of grapevine. Mealybugs overwinter under bark and move into the grape canopy as the season progresses. Because crawlers are more active than later stages, mealybug movement behavior is likely to be stage specific. To quantify P. ficus demography and movement behavior, a series of laboratory experiments were conducted. First, P. ficus populations were monitored on grapevine seedlings to describe survival, change in size, timing of male pupation, and timing of oviposition over a 6-wk period. Subsequently, cohorts of mealybugs were generated by infesting grapevines with crawlers and holding infested grapevines for a specified duration of 0 (crawlers), 1, 2, 3, or 4 wk. Crawlers (0-wk) were more likely to move upwards and towards a light source, than all other age cohorts tested. Further, mealybugs from 4-wk-old cohorts were more likely to move downward than all other age cohorts tested. Results suggest that crawlers are more likely to move to the top of grapevines by moving upwards and orienting towards either the sun or the moon than all other age cohorts tested, whereas older gravid females are more likely to move downward. Passive movement of mealybugs on farm machinery or animals requires surviving a host free period. To quantify risk of passive movement, establishment rates and effects of starvation on each age cohort were quantified. Larger and older mealybugs were more likely to establish on grapevines than smaller and younger mealybugs. Further, mealybug longevity in absence of food was greater for older cohorts compared to younger cohorts. Crawlers survived an average of 2 days without food, whereas females from 4-wk-old cohorts survived for an average of 11 days without food. Further, 70% of starved females from 4-wk-old cohorts deposited fertile eggs. In the absence of food, some mealybugs from cohorts aged 2-, 3-, and 4-wk formed pupa with viable males emerging. Adult males from starved nymphs lived for an average of 3 days post-emergence. Results provide methods for producing cohorts of mealybugs of predictable size and stage and provides insight into P. ficus demography and movement behavior.

Males That Silence Their Father's Genes: Genomic Imprinting of a Complete Haploid Genome.

Genetic conflict is considered a key driver in the evolution of reproductive systems with non-Mendelian inheritance, where parents do not contribute equally to the genetic makeup of their offspring. One of the most extraordinary examples of non-Mendelian inheritance is paternal genome elimination (PGE), a form of haplodiploidy which has evolved repeatedly across arthropods. Under PGE, males are diploid but only transmit maternally inherited chromosomes, while the paternally inherited homologues are excluded from sperm. This asymmetric inheritance is thought to have evolved through an evolutionary arms race between the paternal and maternal genomes over transmission to future generations. In several PGE clades, such as the mealybugs (Hemiptera: Pseudococcidae), paternal chromosomes are not only eliminated from sperm, but also heterochromatinized early in development and thought to remain inactive, which could result from genetic conflict between parental genomes. Here, we present a parent-of-origin allele-specific transcriptome analysis in male mealybugs showing that expression is globally biased toward the maternal genome. However, up to 70% of somatically expressed genes are to some degree paternally expressed, while paternal genome expression is much more restricted in the male reproductive tract, with only 20% of genes showing paternal contribution. We also show that parent-of-origin-specific gene expression patterns are remarkably similar across genotypes, and that genes with completely biparental expression show elevated rates of molecular evolution. Our results provide the clearest example yet of genome-wide genomic imprinting in insects and enhance our understanding of PGE, which will aid future empirical tests of evolutionary theory regarding the origin of this unusual reproductive strategy.

No evidence for an intragenomic arms race under paternal genome elimination in Planococcus mealybugs.

Genomic conflicts arising during reproduction might play an important role in shaping the striking diversity of reproductive strategies across life. Among these is paternal genome elimination (PGE), a form of haplodiploidy which has independently evolved several times in arthropods. PGE males are diploid but transmit maternally inherited chromosomes only, whereas paternal homologues are excluded from sperm. Mothers thereby effectively monopolize the parentage of sons, at the cost of the father's reproductive success. This creates striking conflict between the sexes that could result in a co-evolutionary arms race between paternal and maternal genomes over gene transmission, yet empirical evidence that such an arms race indeed takes place under PGE is scarce. This study addresses this by testing whether PGE is complete when paternal genotypes are exposed to divergent maternal backgrounds in intraspecific and hybrid crosses of the citrus mealybug, Planococcus citri, and the closely related Planococcus ficus. We determined whether males can transmit genetic information through their sons by tracking inheritance of two traits in a three-generation pedigree: microsatellite markers and sex-specific pheromone preferences. Our results suggest leakages of single paternal chromosomes through males occurring at a low frequency, but we find no evidence for transmission of paternal pheromone preferences from fathers to sons. The absence of differences between hybrid and intraspecific crosses in leakage rate of paternal alleles suggests that a co-evolutionary arms race cannot be demonstrated on this evolutionary timescale, but we conclude that there is scope for intragenomic conflict between parental genomes in mealybugs. Finally, we discuss how these paternal escapes can occur and what these findings may reveal about the evolutionary dynamics of this bizarre genetic system.

The Effects of Endophytic Beauveria bassiana Inoculation on Infestation Level of Planococcus ficus, Growth and Volatile Constituents of Potted Greenhouse Grapevine (Vitis vinifera L.).

Endophytic entomopathogenic fungi are being explored for the management of phytophagous insect pests. The effects of Beauveria bassiana (Hypocreales) inoculation of grape plants on the infestation level of P. ficus, tissue nutrient contents, and growth and volatile constituents of potted grape plants were assessed. Grapevine plants were individually inoculated with a suspension of 1 × 108 conidia mL-1 of B. bassiana by drenching before experimentally infesting each of them with thirty adult females of P. ficus. At four weeks post-treatment, the fungus was re-isolated from leaves of 50% of the fungus-exposed plants. However, no significant difference (p > 0.05) was observed in all the plant growth parameters measured in the fungus-treated and control plants. Plant tissue analysis revealed markedly higher contents of calcium (Ca) and magnesium (Mg) in the leaf tissue of plants exposed to the B. bassiana relative to the control. Gas chromatography mass spectrometry (GC-MS) analyses showed that a significantly (X² = 5.1; p < 0.02) higher number of known anti-insect volatile compounds (nine) were present among fungus treated plants compared to the control plants (five). Naphthalene, which is toxic to insects and humans, was detected only in the volatiles of the fungus-exposed plants. B. bassiana did not have any significant effect on total polyphenol, alkaloid, and flavonoids. Overall, treatment with fungus did not inhibit the infestation by P. ficus. In conclusion, these findings shed light on some of the mechanisms involved in endophytic fungus-plant-insect interactions.

Systemic Insecticide Applications: Effects on Citrus Mealybug (Hemiptera: Pseudococcidae) Populations Under Greenhouse Conditions.

The citrus mealybug, Planococcus citri (Risso) (Hemiptera: Pseudococcidae), is a major insect pest of greenhouse-grown horticultural crops. Applications of systemic insecticides as drenches to the growing medium are typically used by greenhouse producers to prevent or suppress citrus mealybug populations. A comprehensive study was conducted that included 11 experimental trials designed to assess the effects of growing medium applications of six systemic insecticides (azadirachtin, dinotefuran, flonicamid, imidacloprid, spirotetramat, and thiamethoxam) in preventing the establishment of and suppressing citrus mealybug populations on different plant species. The experimental trials included four plant species: Solenostemon scutellarioides, Gerbera jamesonii, Begonia × semperflorens-cultorum, and Salvia splendens, with six different cultivars grown in research greenhouses similar to those used in greenhouse production systems. In addition, feeding location (plant stem, leaf top, and leaf bottom) of citrus mealybugs on the various plants was also recorded. Overall, percent citrus mealybug mortality was consistently <50% (n = 255 to 1,598) for any treatment or rate of application; regardless if the systemic insecticides were applied preventatively or curatively. Percent citrus mealybug mortality did reach 78% (n = 36) for thiamethoxam at 8 times the label rate when plants were treated preventatively. Furthermore, none of the treatments prevented development of citrus mealybug F1 individuals to F2 generation egg-laying females on S. scutellarioides or G. jamesonii plants. Citrus mealybugs varied in their spatial distribution on the plant stem, leaf top, and leaf bottom with no noticeable trends. Therefore, based on the results of the study, systemic insecticides are not effective against the citrus mealybug in greenhouse production systems.

Biparental mealybugs may be more promiscuous than we thought.

Knowledge on the reproductive biology of target insect pest is essential for the effective implementation of pheromone-based pest management tactics. In mealybugs, the second largest family of scale insects, the existence of female multiple mating was recently suggested. In this study, we aimed at testing how general is this behavior in mealybugs, by investigating polygyny and polyandry in two cosmopolitan pest mealybugs, Planococcus citri and Pseudococcus calceolariae. Males of these species were able to mate an average of 11.9 and 13.3 females, respectively, during their lifespan. The number of fertilized females per male decreased with male age/mating history for both mealybugs. We found no differences in female fecundity and fertility, when fertilized by males with different mating history. When we used male age as a proxy of mating history, we observed a significant negative effect on female fecundity. The females of both species remained receptive after first copula and eventually mated multiple times. The percentage of remated females of P. citri decreased linearly with time since first copula, with some maintaining receptivity up to 24 h. Males showed no preference between virgin and mated females, in static-air olfactometer tests. We found no benefit of female multiple mating in relation to fecundity. In biparental mealybugs, the mating system of males is possibly scramble competition polygyny; and that of females is possibly polyandry, with female receptivity restricted to a relatively short period. We discuss the practical implications of the results for pest management.

Sexual attractiveness and reproductive performance in ageing females of a coccoid insect.

Female age strongly influences reproductive success in various animals. Males are, therefore, expected to show preferential responses to sexual signals released from females of ages suitable for reproduction. Here, however, we report an unexpected and contradictory effect of ageing on sexual attractiveness and reproductive performance in a coccoid insect: the pheromone-based attractiveness of females increased with ageing, though their reproductive performance was in rapid decline. Surprisingly, senescent females continued releasing relatively high amounts of pheromone and maintained their sexual attractiveness, even at ages when they had almost completely lost fertility, with reduced densities of endosymbionts to support their physiology. Our dataset suggests a potential sexual conflict within a pheromone communication system, where females benefit at males' expense through deceptive signals of fertility.

Determining the geographic origin of invasive populations of the mealybug Planococcus ficus based on molecular genetic analysis.

Determining the most likely source of an invasive pest species might help to improve their management by establishing efficient quarantine measures and heading the search of efficient biological control agents. Planococcus ficus is an invasive mealybug pest of vineyards in Argentina, California, Mexico, Peru and South Africa. This mealybug pest had a previously known geographic distribution spanning southern Europe, the Middle East, and parts of northern Africa. In North America, Pl. ficus was first discovered in the early 1990s and soon thereafter in Mexico. To determine the origin of invasive populations in North America, Pl. ficus from California and Mexico were compared with material throughout its presumptive native range in the Mediterranean region, as well as material collected from an older invasion in South Africa and recently invaded Argentina. From each sample location, genomic DNA was sequenced for the nuclear internal transcribed spacer one (ITS1) and the mitochondrial cytochrome c. oxidase one (CO1). Phylogenetic analyses of CO1, ITS1 and concatenated CO1 and ITS1 data-sets using Bayesian and neighbor-joining analysis support two major divisions: a European grouping (Europe, Tunisia, Turkey) and a Middle Eastern grouping (Israel and Egypt). The invasive populations in Argentina and South Africa align with the European group and the invasive populations in North America align with the Middle Eastern group, with one Israel sample aligning closely with the North American clade, suggesting that Israel was the origin of those populations.

Herbivory-induced maternal effects on growth and defense traits in the clonal species Alternanthera philoxeroides.

Plants have evolved a variety of defense traits against foliar herbivory, including the production of primary and secondary metabolites, the allocation of chemical compounds, and morphological plasticity. Using two vegetative generations of the invasive clonal species Alternanthera philoxeroides, we investigated the effects of maternal and offspring herbivory by Planococcus minor on the integrative defense strategy of plants. Herbivory severely inhibited leaf, stolon and root growth, as well as the production of primary metabolites (soluble sugars, starch, and total non-structural carbohydrates in stolons), and decreased average leaf area and specific leaf area of the second-generation A. philoxeroides. The changes in growth measures of the first-generation A. philoxeroides with herbivory were consistent with that of the second generation. By contrast, herbivory basically did not affect the concentration of non-structural carbohydrate compounds in the roots, and even increased the concentrations of N and total phenols in taproots. Furthermore, herbivory-induced maternal effects also reduced the growth of the second-generation plants. The results suggest that A. philoxeroides is capable of adapting to herbivory by P. minor, mainly via the allocation of available resources in belowground organs, and that the herbivory effect can persist across vegetative generations. These features may potentially improve the regeneration and tolerance of A. philoxeroides after a short-term herbivory.

Repeated replacement of an intrabacterial symbiont in the tripartite nested mealybug symbiosis.

Stable endosymbiosis of a bacterium into a host cell promotes cellular and genomic complexity. The mealybug Planococcus citri has two bacterial endosymbionts with an unusual nested arrangement: the γ-proteobacterium Moranella endobia lives in the cytoplasm of the ß-proteobacterium Tremblaya princeps These two bacteria, along with genes horizontally transferred from other bacteria to the P. citri genome, encode gene sets that form an interdependent metabolic patchwork. Here, we test the stability of this three-way symbiosis by sequencing host and symbiont genomes for five diverse mealybug species and find marked fluidity over evolutionary time. Although Tremblaya is the result of a single infection in the ancestor of mealybugs, the γ-proteobacterial symbionts result from multiple replacements of inferred different ages from related but distinct bacterial lineages. Our data show that symbiont replacement can happen even in the most intricate symbiotic arrangements and that preexisting horizontally transferred genes can remain stable on genomes in the face of extensive symbiont turnover.

Diel periodicity of pheromone release by females of Planococcus citri and Planococcus ficus and the temporal flight activity of their conspecific males.

The diel periodicity of sex pheromone release was monitored in two mealybug species, Planococcus citri and Planococcus ficus (Hemiptera; Pseudococcidae), using sequential SPME/GCMS analysis. A maximal release of 2 ng/h pheromone by 9-12-day-old P. citri females occurred 1-2 h before the beginning of photophase. The highest release of pheromone by P. ficus females was 1-2 ng/2 h of 10-20-day-old females, approximately 2 h after the beginning of photophase. Mating resulted in termination of the pheromone release in both mealybug species. The temporal flight activity of the males was monitored in rearing chambers using pheromone baited delta traps. Males of both P. citri and P. ficus displayed the same flight pattern and began flying at 06:00 hours when the light was turned on, reaching a peak during the first and second hour of the photophase. Our results suggest that other biparental mealybug species display also diel periodicities of maximal pheromone release and response. Direct evaluation of the diel periodicity of the pheromone release by the automatic sequential analysis is convenient and will be very helpful in optimizing the airborne collection and identification of other unknown mealybug pheromones and to study the calling behavior of females. Considering this behavior pattern may help to develop more effective pheromone-based management strategies against mealybugs.

Estimation of stage duration distributions and mortality under repeated cohort censuses.

Many processes in nature can be viewed as arising from subjects progressing through sequential stages and may be described by multistage models. Examples include disease development and the physiological development of plants and animals. We develop a multistage model for sampling designs where a small set of subjects is followed and the number of subjects in each stage is assessed repeatedly for a sequence of time points, but for which the subjects cannot be identified. The motivating problem is the laboratory study of developing arthropods through stage frequency data. Our model assumes that the same individuals are censused at each time, introducing among sample dependencies. This type of data often occur in laboratory studies of small arthropods but their detailed analysis has received little attention. The likelihood of the model is derived from a stochastic model of the development and mortality of the individuals in the cohort. We present an MCMC scheme targeting the posterior distribution of the times of development and times of death of individuals. This is a novel type of MCMC that uses customized proposals to explore a posterior with disconnected support arising from the fact that individual identities are unknown. The MCMC algorithm may be used for inference about parameters governing stage duration distributions and mortality rates. The method is demonstrated by fitting the development model to stage frequency data of a mealybug cohort placed on a grape vine.

How does Tremblaya princeps get essential proteins from its nested partner Moranella endobia in the Mealybug Planoccocus citri?

Many insects maintain intracellular mutualistic symbiosis with a wide range of bacteria which are considered essential for their survival (primary or P-endosymbiont) and typically suffer drastic genome degradation. Progressive loss of P-endosymbiont metabolic capabilities could lead to the recruitment of co-existent facultative endosymbiont (secondary or S-endosymbiont), thus adding more complexity to the symbiotic system. Planococcus citri, among other mealybug species, harbors an unconventional nested endosymbiotic system where every Tremblaya princeps cell (ß-proteobacterium) harbors many Moranella endobia cells (γ-proteobacterium). In this system, T. princeps possess one of the smallest prokaryote genome known so far. This extreme genome reduction suggests the supply of many metabolites and essential gene products by M. endobia. Although sporadic cell lysis is plausible, the bacterial participation on the regulation of the predicted molecular exchange (at least to some extent) cannot be excluded. Although the comprehensive analysis of the protein translocation ability of M. endobia PCVAL rules out the existence of specific mechanisms for the exportation of proteins from M. endobia to T. princeps, immunolocation of two M. endobia proteins points towards a non-massive but controlled protein provision. We propose a sporadic pattern for the predicted protein exportation events, which could be putatively controlled by the host and/or mediated by local osmotic stress.

Evaluation of efficacy of 18 strains of entomopathogenic nematodes (Rhabditida) against Planococcus citri (Risso, 1813) (Hemiptera: Pseudococcidae) under laboratory conditions.

Planococcus citri (Risso, 1813) (Hemiptera: Pseudococcidae) is an important plant virus vector in grapevine crops in Brazil and other countries. The mealybug grows in roots and leaves of the grapes. Entomopathogenic nematodes (EPNs) are efficient control agents against insects associated to the soil and could be applied with the same equipment used for chemical insecticides. The aim of this study was to select effective EPNs for controlling P. citri females in laboratory conditions (25±1°C, UR 60±10%). We tested 17 native [Steinernema rarum (6 strains), Steinernema glaseri, Steinernema feltiae, Steinernema riobrave, Steinernema sp., Heterorhabditis bacteriophora (7 strains)] and only one exotic strain (Steinernema carpocapsae ALL). The bioassays were done on Petri dishes infested with females of P. citri, which were sprayed with EPNs juveniles. The strain with larger pathogenicity and virulence in laboratory was H. bacteriophora RS33 (from 69.0% to 92.2% of mortality), native of Rio Grande do Sul.

Performance of two isolates of Isaria fumosorosea from hot climate zones in solid and submerged cultures and thermotolerance of their propagules.

Isaria fumosorosea frequently causes mycosis of agricultural pests in the hot semiarid and dry tropical regions of Mexico. Because temperature tolerance restricts the use of fungal biopesticides, we investigated two isolates from these areas for possible development into mycoinsecticides for use in hot weather agricultural zones. We studied the effects of culture system (solid or submerged cultures) and temperature on the fungal growth, extracellular enzyme production, pathogenicity, and thermotolerance of the produced propagules. Between 20 and 28 °C, the specific growth rates of the isolate PCC were higher on solid media, but in the submerged culture, the isolate P43A grew faster even at temperatures of up to 34 °C. On solid media, P43A produced 1.5-fold more proteases than PCC, but in the submerged culture, both strains had similar activities. Under the same culture conditions, PCC produced a blastospore:conidia ratio of 1:2, and P43A produced a ratio of 1:5. PCC aerial conidia had the shortest Lethal Time 50 (LT(50), the time to reach 50 % mortality) against Galleria mellonella larvae, but LT(50) was equal for the aerial conidia and the submerged propagules of P43A and PCC. The submerged and aerial propagules of P43A were more thermotolerant than those of PCC. Each isolate performed differently in each culture system, and we concluded that the intended production method should be included as a criterion for screening of entomopathogenic fungus. We found that thermotolerance is a specific characteristic of an isolate from a given species. Because of its specific characteristics, P43A shows more promise for the development of a submerged conidia-based mycoinsecticide for foliar application in aqueous form in hot climate regions.

Insecticidal activity of plant essential oils against the vine mealybug, Planococcus ficus.

The vine mealybug, Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae), is a pest in grape vine growing areas worldwide. The essential oils from the following aromatic plants were tested for their insecticidal activity against P. ficus: peppermint, Mentha piperita L. (Lamiales: Lamiaceae), thyme-leaved savory, Satureja thymbra L., lavender, Lavandula angustifolia Mill, and basil, Ocimum basilicum L. Essential oils from peels of the following fruits were also tested: lemon, Citrus limon L. (Sapindales: Rutaceae), and orange, C. sinensis L. The reference product was paraffin oil. Bioassays were conducted in the laboratory by using spray applications on grape leaves bearing clusters of P. ficus of one size class, which mainly represented either 3rd instar nymphs or pre-ovipositing adult females. The LC50 values for each essential oil varied depending on the P. ficus life stage but did not significantly differ between 3(rd) instar nymphs and adult females. The LC50 values of the citrus, peppermint, and thyme-leaved savory essential oils ranged from 2.7 to 8.1 mg/mL, and the LC50 values of lavender and basil oil ranged from 19.8 to 22.5 and 44.1 to 46.8 mg/mL, respectively. The essential oils from citrus, peppermint and thymeleaved savory were more or equally toxic compared to the reference product, whereas the lavender and basil essential oils were less toxic than the paraffin oil. No phytotoxic symptoms were observed on grape leaves treated with the citrus essential oils, and low phytotoxicity was caused by the essential oils of lavender, thyme-leaved savory, and mint, whereas the highest phytotoxicity was observed when basil oil was used.

Potential of South African entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) for control of the citrus mealybug, Planococcus citri (Pseudococcidae).

Planococcus citri, the citrus mealybug, is the most important species of mealybug known to infest citrus in South Africa. Various laboratory bioassays were conducted to determine the potential of entomopathogenic nematodes to control P. citri. Adult female P. citri were screened for susceptibility to six indigenous nematode species. P. citri was found to be most susceptible to Steinernema yirgalemense and Heterorhabditis zealandica, causing 97% and 91% mortality, respectively. The development of nematodes after infecting adult female P. citri showed both H. zealandica and S. yirgalemense were able to complete their life cycles inside the host. Further bioassays illustrated a linear relationship between mealybug mortality and the concentration of nematodes applied, with the highest level of control using a concentration of 80 infective juveniles (IJs)/insect. As nematodes would be used as an above-ground application to control P. citri in citrus orchards, available water is a major limiting factor. Insecticidal activity proved to be dependent on the available surface moisture after nematode application. The water activity (a(w)) bioassay indicated that S. yirgalemense to be two times more tolerant to lower levels of free water, with a(w50)=0.96 and a(w90)=0.99, compared to H. zealandica with a(w50)=0.98 and a(w)90=1.0. After application, nematodes have a limited time frame in which to locate and infect hosts, as the level of available free water gradually decreases, as trees dry out. S. yirgalemense proved able to locate and infect P. citri quicker than H. zealandica. Nematode activity was not significantly affected when exposed to 15°C, 20°C and 25°C. IJs were able to infect P. citri at an exposure time as short as half an hour. Results also showed that the first 2-4h post application is the most decisive time for establishing successful infection of mealybugs. This is the first report on the potential use of nematodes for the control of P. citri.

Endocrocin and its derivatives from the Japanese mealybug Planococcus kraunhiae.

The Japanese mealybug, Planococcus kraunhiae, is suitable as a model insect for biosynthetic studies on mealybug pigments. Four yellow pigments, including two novel ones, were isolated from the mealybug bodies and characterized as endocrocin, a dicarboxylic acid named fujikonaic acid (1), emodin 1-O-ß-D-glucopyranoside and 7-hydroxyemodin 1-O-ß-D-glucopyranoside (2). The enzymatic activity of emodin 1-O-glucosyltransferase was observed in the extracts of insect bodies.