Pheromone-baited traps for assessment of seasonal activity and population densities of mealybug species (Hemiptera: Pseudococcidae) in nurseries producing ornamental plants

Operational parameters of traps baited with the pheromones of three mealybug species were optimized in nurseries producing ornamental plants. All pheromone doses (1-320 microg) attracted Pseudococcus longispinus (Targioni Tozzetti) and Pseudococcus viburni (Signoret) males, with the lowest dose (1 microg) attracting the fewest males for both species. Doses of 3.2-100 microg were as attractive to male P. longispinus as the highest dose (320 microg); doses from 10 to 320 microg were equally attractive for P. viburni males. Lures containing 25-microg doses of either pheromone had effective field lifetimes of at least 12 wk. Experiments were performed to test the efficacy of combining multiple pheromones to attract several species of mealybugs simultaneously. Lures loaded with a mixture of the pheromones of P. longispinus, P. viburni, and Planococcus citri (Risso) were as attractive to P. viburni and P. citri as lures with their individual pheromones. Response of P. longispinus to the blend was decreased by 38% compared with its pheromone as a single component. A subsequent trial with two-component blends showed that the pheromone ofP. citri was responsible for this modest decrease in P. longispinus response. This should not affect the overall efficacy of using these lures for monitoring the presence of all three mealybug species simultaneously. Pheromone traps were used to detect infestations of P. longispinus throughout the season and to track population cycles. When pheromone-baited traps for P. longispinus were compared with manual sampling, trap counts of male mealybugs were significantly correlated with mealybugs counted on plants in the vicinity of the traps.     

Development and optimization of methods for using sex pheromone for monitoring the mealybug Planococcus ficus (Homoptera: Pseudococcidae) in California vineyards

The sex pheromone of the vine mealybug Planococcus ficus Signoret has been identified as a single component, lavandulyl senecioate. Racemic lavandulyl senecioate was as attractive to male mealybugs as the insect-produced (S)-enantiomer, indicating that the unnatural enantiomer is not inhibitory. Lavandulol, which also was found in extracts from virgin females, antagonized attraction of males at higher doses. Rubber septum lures loaded with 10- to 1,000-microg doses of the pheromone were equally attractive, and lures loaded with 100 microg of racemic pheromone remained attractive for at least 12 wk under field conditions. Delta traps were more effective than double-sided sticky cards and minimized captures of nontarget insects. Pheromone-baited traps had an effective range of at least 50 m. Comparison of visual sampling methods and sampling of males with pheromone-baited traps revealed that trap catches were significantly correlated with the results from visual sampling methods, and with economic damage.     

Diagnostic markers for Planococcus ficus (Signoret) and Planococcus citri (Risso) by random amplification of polymorphic DNA-polymerase chain reaction and species-specific mitochondrial DNA primers

Abstract:  Planococcus ficus (Signoret) and Planococcus citri (Risso) (Hom., Pseudococcidae) are important phytophagous components in different agroecosystems. The two species may coexist in the same environment and are most difficult to distinguish by morphological features. The aim of this study was to find genetic markers suitable for distinguishing P. ficus from P. citri , to assist in the rapid identification of field specimens. By using synthetic sex pheromone-baited traps, pure male populations of both species were collected from a vineyard and from a citrus orchard in northern Sardinia, Italy. Individual males of citrus and vine mealybugs were preliminarily examined by the random amplification of polymorphic DNA (RAPD) technique. Among twelve 10-mer random primers, the oligonucleotide OPL-12 generated several markers suitable for distinguishing between the two species. This primer was then used to characterize individual males and females of both mealybug species collected near pheromone-baited traps in vineyards and orange orchards from different geographic areas. Reference samples from other regions of southern Italy were also included. A clear differentiation of the two species was accomplished according to their pattern of amplification, thus confirming a high level of intra-specific genetic homogeneity. Consequently, two fragments of the cytochrome c oxidase I gene from P. citri and P. ficus were compared and two pairs of species-specific polymerase chain reaction (PCR) primers were developed based on diverging sequences. These primers allowed sensitive and reliable PCR identification of both males and females of P. citri and of P. ficus of different geographic origin.     

A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae)

Molecular species identification is becoming more wide-spread in diagnostics and ecological studies, particularly with regard to insects for which morphological identification is difficult or time-consuming. In this study, we describe the development and application of a single-step multiplex PCR for the identification of three mealybug species (Hemiptera: Pseudococcidae) associated with grapevine in South Africa: Planococcus ficus (vine mealybug), Planococcus citri (citrus mealybug) and Pseudococcus longispinus (longtailed mealybug). Mealybugs are pests on many commercial crops, including grapevine, in which they transmit viral diseases. Morphological identification of mealybug species is usually time-consuming, requires a high level of taxonomic expertise and usually only adult females can be identified. The single-step multiplex PCR developed here, based on the mitochondrial cytochrome c oxidase subunit 1 (CO I) gene, is rapid, reliable, sensitive, accurate and simple. The entire identification protocol (including DNA extraction, PCR and electrophoresis) can be completed in approximately four hours. Successful DNA extraction from laboratory and unparasitized field-collected individuals stored in absolute ethanol was 97%. Specimens from which DNA could be extracted were always correctly identified (100% accuracy). The technique developed is simple enough to be implemented in any molecular laboratory. The principles described here can be extended to any organism for which rapid, reliable identification is needed.     

Molecular and Morphological Identification of Mealybug Species (Hemiptera: Pseudococcidae) in Brazilian Vineyards

Mealybugs (Hemiptera: Pseudococcidae) are pests constraining the international trade of Brazilian table grapes. They damage grapes by transmitting viruses and toxins, causing defoliation, chlorosis, and vigor losses and favoring the development of sooty mold. Difficulties in mealybug identification remain an obstacle to the adequate management of these pests. In this study, our primary aim was to identify the principal mealybug species infesting the major table grape-producing regions in Brazil, by morphological and molecular characterization. Our secondary aim was to develop a rapid identification kit based on species-specific Polymerase Chain Reactions, to facilitate the routine identification of the most common pest species. We surveyed 40 sites infested with mealybugs and identified 17 species: Dysmicoccus brevipes (Cockerell), Dysmicoccus sylvarum Williams and Granara de Willink, Dysmicoccus texensis (Tinsley), Ferrisia cristinae Kaydan and Gullan, Ferrisia meridionalis Williams, Ferrisia terani Williams and Granara de Willink, Phenacoccus baccharidis Williams, Phenacoccus parvus Morrison, Phenacoccus solenopsis Tinsley, Planococcus citri (Risso), Pseudococcus viburni (Signoret), Pseudococcus cryptus Hempel, four taxa closely related each of to Pseudococcus viburni, Pseudococcus sociabilis Hambleton, Pseudococcus maritimus (Ehrhorn) and Pseudococcus meridionalis Prado, and one specimen from the genus Pseudococcus Westwood. The PCR method developed effectively identified five mealybug species of economic interest on grape in Brazil: D. brevipes, Pl. citri, Ps. viburni, Ph. solenopsis and Planococcus ficus (Signoret). Nevertheless, it is not possible to assure that this procedure is reliable for taxa that have not been sampled already and might be very closely related to the target species.     

Attraction of Planococcus ficus males to racemic and chiral pheromone baits: flight activity and bait longevity

The vine mealybug (VM), Planococcus ficus (Signoret), is a worldwide pest and affects mainly grapevine and figs. In Israel, (S)-lavandulyl senecioate [(S)-LS] and (S)-lavandulyl isovalerate [(S)-LI] were identified from VM reared for several generations on potato sprouts, when compared with the identification of only (S)-LS from feral females. Racemic lavandulyl senecioate (LS) can be used for fieldwork because the unnatural enantiomer is benign and does not affect the activity of the pheromone. Lures loaded with 200  μ g of LS remained active for 24 weeks but it is recommended to replace lures after 12 weeks to preserve constant and high attractiveness. Delta sticky traps, baited with 50 or 200  μ g of LS were used to determine the daily flight pattern and the seasonal flight activity including vine plant infestation. A positive correlation between peak mealybug density and male capture was observed strengthening the usefulness of the pheromone traps for monitoring the pest. Addition of racemic or the two enantiomers of lavandulyl isovalerate (LI) to LS decreased the capture of VM males. The level of inhibition varied during 2003–2004. Both enantiomers of LI displayed the same antagonistic activity. Addition of (S)-lavandulol to the pheromone resulted in shut-down of the traps. Inhibition by the antipode, (R)-lavandulol, was moderate indicating a certain degree of chiral discrimination.     

Hot-water treatments for control of Planococcus ficus (Homoptera: Pseudococcidae) on dormant grape cuttings

Hot-water immersions were tested for control of mealybug Planococcus ficus (Signoret), on dormant grape cuttings used for nursery stock. A range of hot-water temperatures (47-58 degrees C) were evaluated at immersion periods of 2, 5, 10, or 20 min, by using a total of 353,720 mealybugs across all treatments. A 5-min immersion at 51 degrees C is effective in killing > 99% of P. ficus. At or above this immersion period and temperature, there was no difference in mealybug stage mortality. We evaluated a commercial operation, which used a 5-min immersion in each of three water tanks: preheating (30.0 +/- 3 degrees C), hot-water (52.8 +/- 0.3 degrees C), and cooling (23 +/- 3 degrees C). The commercial procedure provided 99.8-100% mealybug control in each of three separate trials.     

Responses of Second-Instar Male Nymphs of Four Mealybug Species (Hemiptera: Pseudococcidae) to Conspecific and Heterospecific Female Sex Pheromones

The response of the late second-instar male nymphs of the mealybug species (Hemiptera, Pseudococcidae), Planococcus citri (Risso), Planococcus ficus (Signoret), Pseudococcus cryptus Hempel Nipaecoccus viridis (Newstead), to their conspecific and heterospecific female pheromone was studied. Males that exhibited the typical appearance of late-second-instar nymphs were tested. The male behavior was monitored soon after their exposure to the tested female sex pheromone in glass Petri dish arenas. Male nymph behavior toward the pheromone source was characterized based on their aggregation on the disks in the arena. Males of all four tested mealybug species were attracted to their conspecific female pheromone. By contrast, almost no interceptions of male nymphs with disks impregnated with a heterospecific female pheromone were observed. The mode of attraction of each of male nymphs of P. ficus, among most of the tested individuals (>80%), to the conspecific female sex pheromone, (S)-lavandulyl senecioate and or (S)-lavandulyl isovalerate, was the same as the mode of attraction latter on as adult. We suggest that by being attracted to the conspecific pheromone these males may direct themselves to a suitable pupation site near conspecific non-sibling mature females, thus preventing inbreeding. The repellency of heterospecific sex pheromone to males that are looking for a pupation site suggests that the latter try to avoid close contact with heterospecific females.     

利用种特异性COI引物（SS-COI）鉴别扶桑绵粉蚧

扶桑绵粉蚧Phenacoccus solenopsis Tinsley是我国近年新发现的一种严重威胁农林业生产的重要外来入侵害虫。针对扶桑绵粉蚧与其他粉蚧类昆虫难以准确快速识别且适生区广泛的问题, 以扶桑绵粉蚧为靶标, 以我国常见的其他7种粉蚧为参照, 采用基于线粒体DNA细胞色素C氧化酶亚基Ⅰ (mtDNA COI) 基因序列的种特异性(species-specific COI, SS-COI) PCR方法, 研究其快速分子检测技术。通过已知粉蚧的COI基因序列设计通用型引物1对, 获得扶桑绵粉蚧及其他7种粉蚧包括康氏粉蚧Pseudococcus comstocki Kuwana、 南洋臀纹粉蚧Planococcus lilacius Cockerell、 木槿曼粉蚧Maconellicoccus hirsutus (Green)、 甘蔗红粉蚧Saccharicoccus sacchari (Cockerell)、 新菠萝灰粉蚧Dysmicoccus neobrevipes Beardsley、 番石榴粉蚧Planococcus minor Maskel和石蒜绵粉蚧Phenacoccus solani Ferris的COI基因序列, 根据测序结果及数据库中已知粉蚧的COI基因序列设计SS COI引物1对(PSZTF1/PSZTR1), 其扩增片段大小为546 bp。种特异性检验结果表明, 该引物只对扶桑绵粉蚧的COI基因具有扩增能力, 对其他7种粉蚧不具有扩增效果; 该引物不仅对成虫具有良好的扩增能力, 对不同虫态的扶桑绵粉蚧以及来自我国不同省市的14个地理种群和口岸截获的来自巴基斯坦的扶桑绵粉蚧亦具有同样的扩增效能。这些结果表明, 该技术体系完全可用于扶桑绵粉蚧的准确识别及其检测监测, 对有效阻截其进一步扩张蔓延意义重大。     

Defense Response of Native and Alien Mealybugs (Hemiptera: Pseudococcidae) Against the Solitary Parasitoid Anagyrus sp. nr. pseudococci (Girault) (Hymenoptera: Encyrtidae)

The host behavioral and immune (encapsulation) defenses against the parasitoid Anagyrus sp. nr. pseudococci were compared for five mealybug species with different phylogenetic relationships and geographical origins: i) a Mediterranean native mealybug species, Planococcus ficus, with a long co-evolutionary history with the parasitoid; ii) three alien mealybugs species, Planococcus citri, Pseudococcus calceolariae and Pseudococcus viburni, with a more recent co-evolutionary history; and iii) a fourth alien mealybug species, Phenacoccus peruvianus, with no previous common history with the parasitoid. Three host defense behaviors were registered: abdominal flipping, reflex bleeding and walking away. The native host Pl. ficus and its congener Pl. citri exhibited the lowest probability of defense behavior (0.11?±?0.01 and 0.09?±?0.01 respectively), whereas the highest value was observed in P. viburni (0.31?±?0.02). Intermediate levels of defense behavior were registered for Ps. calceolariae, and Ph. peruvianus. The probability of parasitoid encapsulation was lowest and highest for two alien host species, Ph. peruvianus (0.20?±?0.07) and Ps. viburni (0.86?±?0.05), respectively. The native host Pl. ficus, its congener Pl. citri and Ps. calceolariae showed intermediate values (0.43?±?0.07, 0.52?±?0.06, and 0.45?±?0.09, respectively). The results are relevant with respect to biological control and to understand possible evolutionary processes involved in host range of A. sp. nr. pseudococci.     

Suitability of five mealybug species (Hemiptera,Pseudococcidae) as hosts for the solitary parasitoid Anagyrus sp. nr. pseudococci (Girault) (Hymenoptera: Encyrtidae)

Anagyrus sp. nr. pseudococci is an endoparasitoid which has been used as a biological control agent of mealybug pests. In this study, we compared the suitability of five mealybugs species with different phylogenetic relationships and geographical origins as hosts of this parasitoid. The selected mealybugs were: (1) a Mediterranean-native species, Planococcus ficus, sharing a long co-evolutionary history with the parasitoid; (2) three exotic species, the Afrotropical Planococcus citri, the Australasian Pseudococcus calceolariae and the Neotropical Pseudococcus viburni, with a recent history; and (3) the Neotropical Phenacoccus peruvianus, with no previous common history with the parasitoid. Host suitability was assessed based on different fitness parameters, such as body size, developmental time, emergence rate and sex ratio. The parasitoid was able to complete development in all mealybug species. Nevertheless, its emergence rate significantly varied among mealybug species, with the highest values observed in Pl. ficus and Pl. citri, intermediate values in Ps. calceolariae and the lowest ones in Ps. viburni and Ph. peruvianus. The body size of adult wasp females varied with host suitability and was positively correlated with other measures of parasitoid fitness, including the emergence rate and the sex ratio. The parasitoid developmental time differed among mealybug species but did not correlate with any other measure of fitness. A female biased sex ratio was found in the parasitoid progeny emerged from all mealybug species, except in Ps. viburni and Ph. peruvianus. There was a direct relationship between the proportion of females in the parasitoid progeny and the emergence rate.     

Molecular-based approach to the differentiation of mealybug (Hemiptera: Pseudococcidae) species.

The rDNA internal transcribed spacer (ITS) region of 4 mealybug species, Pseudococcus viburni (Signoret), P. longispinus (Targiono-Tozzetti), P. calceolariae (Maskell), and P. similans (Lidgett), was isolated by polymerase chain reaction (PCR) amplification, cloned, and sequenced. In this region of the genome there were numerous differences, including nucleotide substitutions, insertions, or deletions between P. viburni, P. longispinus, and P. calceolariae, whereas P. calceolariae and P. similans were very similar. Based on sequence differences between the ITS regions, we designed PCR primers that were able to differentiate the 4 mealybug species and that correlated with morphological differences found between adult females of these species. The PCR amplification by using the species-specific primers enabled the differentiation of not only adult females but also eggs, juveniles, and adult males, which was not previously possible by using conventional identification methods.     

Pheromone-based mating disruption of Planococcus ficus (Hemiptera: Pseudococcidae) in California vineyards

Experiments were conducted to test a mating disruption program for the mealybug Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) in California vineyards. The sprayable, microencapsulated formulation of the racemic sex pheromone lavandulyl senecioate was applied with an air-blast sprayer, using three and four applications in 2003 and 2004, respectively. Mating disruption was combined with an application of buprofezin (2004) in June. Compared with a no-pheromone control, there were significantly lower season-long trap catches of adult males, season-long mealybug densities (2003 only), and crop damage in mating disruption plots. The amount of mealybug reduction and mechanisms that resulted in lower crop damage in mating disruption plots is discussed. In samples taken during the growing season (April to September), mealybug density was only 12.0 +/- 15.6 and 31.1 +/- 11.6% lower in the mating disruption plots than in control plots in 2003 and 2004, respectively. In the mating disruption treatment, mealybug egg production was significantly lower (2003 only), as were the proportion of ovisacs and crawlers produced. There was no treatment impact on percentage of parasitism. Mealybug density influenced treatment impact. In 2004, vines were categorized as having low, medium, or high mealybug densities during a preapplication survey. After treatment application, mealybug density was reduced by 86.3 +/- 6.3% on vines in the low mealybug density category, but it was unchanged on vines in the high density category. Another factor that reduced treatment impact was the relatively short effective lifetime of the sprayable formulation.     

中国进境水果携带粉蚧疫情分析(2013—2016)

【目的】中国是水果生产大国和进口大国。水果是最容易携带虫害的产品之一,粉蚧是口岸水果检疫过程中常发现的害虫类群,中国口岸每年从进境水果截获大量粉蚧。通过分析中国进境水果携带粉蚧疫情,可为一线口岸检疫查验提供指导,为相关部门开展产地检疫和口岸监测提供依据,以防止危险性粉蚧的传入,保障中国水果生产安全,促进国际水果贸易健康发展。【方法】通过FAO网站、动植物检验检疫信息资源共享服务平台、中国知网等收集中国进境水果贸易数据及其携带粉蚧疫情,统计分析了中国水果进口贸易情况、粉蚧截获情况以及截获粉蚧的种类、来源地、截获口岸和寄主。【结果】2013—2016年,中国口岸从进境水果上截获的昆虫中粉蚧科昆虫截获量最大,占47.31%;其中截获量前十的粉蚧种类为杰克贝尔氏粉蚧、木槿曼粉蚧、双条拂粉蚧、大洋臀纹粉蚧、南洋臀纹粉蚧、柑橘棘粉蚧、甘蔗簇粉蚧、康氏粉蚧、菠萝灰粉蚧和李比利氏灰粉蚧;而这些有害生物的主要来源地为越南和泰国;粉蚧的主要截获口岸为广西、深圳和云南;而火龙果、榴莲、龙眼和山竹是截获粉蚧最多的进境水果。【结论】中国进境水果粉蚧疫情与水果的贸易量和贸易方式、输出国有害生物发生和检疫除害处理措施、口岸关注度和能力建设等情况相关。     

Inventory and abundance of mealybug species in immature and mature cocoa farms in Côte d'Ivoire

From 2013 to 2018, surveys were conducted in counties not previously surveyed in order to determine species of mealybugs present in the cocoa orchard in Côte d'Ivoire as well as their abundance according to the age of cocoa trees. Immature and mature cocoa trees were inspected to hand‐height in 5 and 29 counties infected with Cacao swollen shoot virus (CSSV). In each cocoa farm, mealybugs were searched for on fruits, leaves, flowers, twigs and trunks. Mealybug species were identified, and colonies were counted. Five mealybug species were identified on immature cocoa trees: Ferrisia virgata, Formicococcus njalensis, Planococcus citri, Planococcus kenyae and Pseudococcus longispinus. In addition to these species, four species, Dysmicoccus brevipes, Maconellicoccus hirsutus, Phenacoccus hargreavesi and Pseudococcus jackbeardsleyi were identified on mature cocoa trees. On immature cocoa trees, Fo. Njalensis, Pl. citri and Ps. longispinus comprised were, respectively, 35%, 33% and 19% of colonies, respectively. On mature cocoa trees, Fo. Njalensis and Pl. citri comprised 63.2% and 21.0%, and others species 15.8%. Nevertheless, the abundance of mealybug species varied according to the age of cocoa trees. The preferred organs of mealybugs were pods (74.1%) followed by twigs (13.4%) and flowers (7.4%). Previously, the mealybug Paracoccus burnerae (Brain) was found on Theobroma cacao, which is the first record for this species in Côte d'Ivoire and on this host‐plant.     

Serological studies on five species of Pseudococcidae (Homoptera)

Abstract. Populations of Planococcus cini, Planococcus ficus, Pseudococcus obscurus, Pseudococcus calceolanae and Pseudococcus longispinus were selected for immunoelectrophoretic tests from which correlation and distance coefficients were calculated. Cluster analysis of serological data allowed construction of a dendrogram in which the phylogenetic relationships among the five species are presented. The conclusions were found to be in agreement with modern views on taxonomic interrelations among the five species based on female and male morphology. The recent separation of Planococcus citn' and Planococcus ficus as independent species is emphasized.     

Pre‐release host range determination of the parasitoid Allotropa burrelli for the biocontrol of Pseudococcus comstocki in France

We performed “no‐choice” tests to study the host range of the parasitoid Allotropa burrelli (Muesebeck) (Hymenoptera: Platygastridae) for use against the Comstock mealybug, Pseudococcus comstocki (Kuwana) (Hemiptera: Pseudococcidae), in Southern France. We tested three Pseudococcidae species as potential non‐target hosts: two species from the same genus (Pseudococcus longispinus and Pseudococcus viburni) and Planococcus citri. Allotropa burrelli did not parasitize any of the non‐target mealybug species tested. No attempt of oviposition was recorded for the three species tested during the first 20 min of parasitoid release and no parasitism occurred in 6–8 hr of exposure of the mealybugs to the parasitoid.     

Hybridization and sex pheromone responses in two closely related mealybug species (Homoptera: Pseudococcidae)

Abstract. Reciprocal crosses were made between the closely related species Planococcus citri and Planococcus ficus by using mating inducers. The pheromone responses of the hybrid offspring were bioassayed. The sex ratio of the offspring showed a striking shift in favour of females in comparison with homospecific matings mainly because of high mortality of male nymphs during the first instar. Hybrid males gave positive reactions only to pheromone extracts from the maternal species and showed no response to extracts from the paternal species. This result may be explained by the lecanoid chromosome system known for mealybug males, in which the paternal set of chromosomes becomes heterochro-matic (condensed and genetically inactive) starting from early embryogeny. Further evidence is, however, presented that (at least in certain tissues) the paternal set reverts to the euchromatic state and to genetical activity.     

Mating disruption method against the vine mealybug,Planococcus ficus: effect of sequential treatment on infested vines

The vine mealybug, Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae), is a major pest of vineyards. Here, we tested the efficacy of the mating disruption method against the pest when applied during one or two successive years in high and low infestation levels. Following 1 year of treatment, at low initial infestation levels a shutdown of pheromone traps was observed, along with a significant reduction in infested vines. With initially high infestation levels, a gradual reduction in infested vines was observed, with a trap shutdown seen only after the second year of pheromone application. We discuss the implications of the male mating disruption method for this pest in which the wingless females are aggregated with limited movement among vines, offering multiple mating opportunities for the flying male.     

Infection dynamics of coexisting beta- and gammaproteobacteria in the nested endosymbiotic system of mealybugs

We investigated the infection dynamics of endosymbiotic bacteria in the developmental course of the mealybugs Planococcus kraunhiae and Pseudococcus comstocki. Molecular phylogenetic analyses identified a betaproteobacterium and a gammaproteobacterium from each of the mealybug species. The former bacterium was related to the beta-endosymbionts of other mealybugs, i.e., "Candidatus Tremblaya princeps," and formed a compact clade in the Betaproteobacteria. Meanwhile, the latter bacterium was related to the gamma-endosymbionts of other mealybugs but belonged to distinct clades in the Gammaproteobacteria. Whole-mount in situ hybridization confirmed the peculiar nested formation in the endosymbiotic system of the mealybugs: the beta-endosymbiont cells were present in the cytoplasm of the bacteriocytes, and the gamma-endosymbiont cells were located in the beta-endosymbiont cells. In nymphal and female development, a large oval bacteriome consisting of a number of bacteriocytes was present in the abdomen, wherein the endosymbionts were harbored. In male development, strikingly, the bacteriome progressively degenerated in prepupae and pupae and became almost unrecognizable in adult males. In the degeneration process, the gamma-endosymbionts disappeared more rapidly than the beta-endosymbionts did. Quantitative PCR analyses revealed that (i) the population dynamics of the endosymbionts in female development reflected the reproductive activity of the insects, (ii) the population dynamics of the endosymbionts were strikingly different between female development and male development, (iii) the endosymbiont populations drastically decreased in male development, and (iv) the gamma-endosymbiont populations decreased more rapidly than the beta-endosymbiont populations in male development. Possible mechanisms underlying the uncoupled regulation of the beta- and gamma-endosymbiont populations are discussed in relation to the establishment and evolution of this unique prokaryote-prokaryote endosymbiotic system.