Naturwissenschaften DOI 10.1007/s00114-014-1218-7
ORIGINAL PAPER
Paternal inheritance in mealybugs (Hemiptera: Coccoidea: Pseudococcidae) Hofit Kol-Maimon & Zvi Mendel & José Carlos Franco & Murad Ghanim
Received: 14 March 2014 / Revised: 2 July 2014 / Accepted: 25 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Mealybugs have a haplodiploid reproduction system, with paternal genome elimination (PGE); the males are diploid soon after fertilization, but during embryogenesis, the male paternal set of chromosomes becomes heterochromatic (HC) and therefore inactive. Previous studies have suggested that paternal genes can be passed on from mealybug males to their sons, but not necessarily by any son, to the next generation. We employed crosses between two mealybug species— Planococcus ficus (Signoret) and Planococcus citri (Risso)— and between two populations of P. ficus, which differ in their mode of pheromone attraction, in order to demonstrate paternal inheritance from males to F2 through F1 male hybrids. Two traits were monitored through three generations: mode of male pheromone attraction (pherotype) and sequences of the internal transcribed spacer 2 (ITS2) gene segment (genotype). Our results demonstrate that paternal inheritance in mealybugs can occur from males to their F2 offspring, through F1 males (paternal line). F2 backcrossed hybrid males expressed paternal pherotypes and ITS2 genotypes although their mother Communicated by: Sven Thatje Electronic supplementary material The online version of this article (doi:10.1007/s00114-014-1218-7) contains supplementary material, which is available to authorized users. H. Kol-Maimon : Z. Mendel : M. Ghanim Department of Entomology, The Volcani Center, ARO, 50250 Bet Dagan, Israel H. Kol-Maimon : Z. Mendel : M. Ghanim (*) Department of Entomology, Robert H. Smith Faculty of Agriculture, Food and Environment, Hebrew University of Jerusalem, POB 12, 76100 Rehovot, Israel e-mail:
[email protected] J. C. Franco Departamento de Ciências e Engenharia de Biossistemas/Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade de Lisboa, 1349-017 Lisboa, Portugal
originated through a maternal population. Further results revealed other, hitherto unknown, aspects of inheritance in mealybugs, such as that hybridization between the two species caused absence of paternal traits in F2 hybrid females produced by F1 hybrid females. Furthermore, hybridization between the two species raised the question of whether unattracted males have any role in the interactions between P. ficus and P. citri. Keywords Reproduction system . Paternal genome elimination . Heterochromatinization . Planococcus citri . Planococcus ficus . Hybridization . Hybrids . Pheromone . ITS2
Introduction The inheritance patterns of traits are influenced by genetic systems, of which scale insects (Hemiptera: Coccoidea) display a remarkable variety (Normark 2004; Ross et al. 2010). The most widespread genetic system among scale insects is paternal genome elimination (PGE) (Bongiorni et al. 1999; Normark 2004; Sanchez 2008; Ross et al. 2010). PGE is a type of haplodiploidy, which is a common genetic system in the animal kingdom, especially among insects (Normark 2003, 2004). Arrhenotokous haplodiploidy, the commonest type among haplodiploid systems, is well known in the Thysanoptera, Hemiptera, Coleoptera, and Hymenoptera (Normark 2003). In arrhenotoky, males develop from unfertilized eggs and possess one set of maternally originated chromosomes (Slobodchikoff and Daly 1971; Cook 1993). However, in PGE, males develop from a diploid zygote consisting of one haploid genome inherited from the mother and another inherited from the father, but in an early embryo stage—soon after fertilization—they lose one set of chromosomes (Normark 2003; Sanchez 2008; Ross et al. 2010). PGE
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is known in Hemiptera, Coleoptera, and Diptera (Normark 2003), and three types are known in scale insects (Ross et al. 2010). The first, termed the lecanoid system, can be found in mealybugs (Pseudococcidae), lac scale insects (Kerriidae), and some felt scales (Eriococcidae). The second was designated after the name of an atypical genus of armored scales, Comstockiella, in which it was found for the first time. The third type termed the diaspidid system is known mostly in armored scales (Diaspididae). In the lecanoid system, the paternal set of chromosomes in males is inactivated during early embryogenesis, remains inactive in the somatic cells, and is then eliminated only during spermatogenesis (Brown and Nur 1964; Nur 1982; Sanchez 2008; Ross et al. 2010). The Comstockiella system is very similar to the lecanoid, except that some chromosomes of the paternal set are lost before spermatogenesis (Ross et al. 2010). Finally, in the diaspidid system, the paternal set of chromosomes in males is completely eliminated during embryogenesis; therefore, the somatic and germ line cells are haploid (Sanchez 2008; Ross et al. 2010). Inactivation of the paternal set of chromosomes in mealybugs is characterized by facultative heterochromatinization (=HC) in contrast to the active maternal euchromatic (=EC) set (Brown and Nur 1964; Bongiorni and Prantera 2003). Heterochromatic chromosomes are condensed, shorter than EC ones, and have no expressed genes (Verma 1990). In mealybugs, HC involves DNA methylation and posttranslational histone modifications (Prantera and Bongiorni 2012). Another case of HC occurs in mammals, where it involves inactivation of the X chromosome (Lyon 1972); only one chromosome is inactivated, and the process also involves DNA methylation and modifications of histones (Heard et al. 2001). However, in mealybugs, the heterochromatic paternal set of male somatic cell chromosomes may later become EC and genetically active in some tissues or organs, such as the midgut, the Malpighian tubules, the salivary glands, the oenocytes, and the serosa, a phenomenon known as reverse HC (Nur 1967). Brown and Nur (1964) found no recombination between HC and EC chromosome sets during spermatogenesis in male mealybug. The fate of chromosomes during the second meiotic division of gametogenesis is completely different between female and male mealybugs (Prantera and Bongiorni 2012): In females, homologous chromosomes undergo crossing over and independent sorting (Ross et al. 2010), whereas in males, in contrast to females, during the second division, the maternal EC set of chromosomes segregates from the paternal HC set. Thus, no crossing over can occur between homologous chromosomes. However, the first meiotic division is equal for males and females, with the separation of sister chromatids creating spermatogonial precursor cells in males (Prantera and Bongiorni 2012). The cytological findings mentioned above (Brown and Nur 1964; Nur 1967; Ross et al. 2010; Prantera and Bongiorni
2012) imply that the paternal set of chromosomes in mealybug males can be expressed in some somatic cells but cannot be passed on to subsequent generations. However, no genetic experiments were reported that concern the fate of paternal traits beyond the first-generation progeny. In the present study, the vine mealybug Planococcus ficus (Signoret) and the citrus mealybug Planococcus citri (Risso) were combined to serve as a working model for revealing the role of paternal inheritance in mealybugs from males to their F2 offspring through F1 males, i.e., the paternal line. P. citri differs taxonomically from P. ficus in several features of female morphology, including the following: the distribution map of dermal pores and ducts and their speciestypical shape, the typical distribution map of setae, and ratios between leg segments (Cox 1981, 1989). Cox (1983) also indicated that, following exposure to extremely high temperatures, P. citri females showed morphological features of P. ficus. Distinction between males of these species by morphological features is based only on the numbers of medial frontal pores (Williams 1985). These two congeners also differ in their development rates (Gray 1954; Gullan and Kosztarab 1997; Walton and Pringle 2005). The P. citri sex pheromone consists of one chemical component (S+)-cis-(1R)-3-isopropenyl-2,2imethylcyclobutanemethanol acetate (=C) (Bier-Leonhardt et al. 1981), whereas in P. ficus, two components are involved, lavandulyl senecioate (=LS) and lavandulyl isovalerate (=LI) (Hinkens et al. 2001; Zada et al. 2003; Kol-Maimon et al. 2010). Attraction of vine mealybug males to LI was documented only among East Mediterranean populations of the vine mealybug but not in the West Mediterranean ones (KolMaimon et al. 2014) or in populations from California (Hinkens et al. 2001). This pattern was manifested in the differing compositions of male pherotypes, i.e., differing modes of male attraction to the two pheromone components, LS and LI, among different geographical populations (KolMaimon et al. 2014). Genetic linkage was demonstrated between the respective pherotypes and development times among P. ficus populations (Kol-Maimon et al. 2010). Sequencing of the nuclear internal transcribed spacer 2 (ITS2) of suspected hybrid specimens is frequently used to demonstrate hybridization and gene flow between various organisms, such as plants (Moody and Les 2002), insects (Stevens et al. 2002; Kol-Maimon et al. 2014), nematodes (La Rosa et al. 2003), and sea stars (Wakabayashi et al. 2008). Additionally, sequencing segments of the ITS2 gene is often used to distinguish between P. citri and P. ficus (Malausa et al. 2010). In the present study, this technique was employed for tracking paternal features among laboratory-induced hybrids. Hybrids of P. citri and P. ficus were first documented in laboratory experiments by Tranfaglia and Tremblay (1982), who showed that the hybrid females displayed intermediate morphological features between their parental species: They
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reported that hybrid males were attracted only to the pheromone extracts of the maternal species. High mortality among hybrid male crawlers was also documented in that study (Rotundo and Tremblay 1982). In a previous study, we used pherotype compositions and ITS2 gene sequences to demonstrate the occurrence of gene flow between P. citri and P. ficus among wild populations (Kol-Maimon et al. 2014), and the results suggested a directional gene flow from P. ficus to P. citri but not vice versa. We further showed that genetic features of P. ficus were present among P. citri populations but not vice versa. Males attracted to LI were also found in P. citri populations from the East Mediterranean but not among those from the West Mediterranean (Kol-Maimon et al. 2014). The main objective of the present study was to investigate the occurrence of paternal inheritance in mealybugs. Two types of crosses were performed to track paternal line inheritance: (i) P. ficus females mated with P. citri males and (ii) P. ficus females of an LI-free (from Portugal) population mated with P. ficus males attracted to an LI (from Israel) population. Two traits were compared between successive generations: (i) mode of male attraction to pheromone (pherotype) and (ii) male and female ITS2 gene sequence (genotype). If paternal inheritance from males to the subsequent generations occurred through their sons (paternal line), then, we would expect to observe paternal traits (P. citri traits and LI attraction) among F1 and F2 hybrid males.
Materials and methods Tested populations and laboratory rearing Crosses were conducted between P. ficus and P. citri, using laboratory-reared individuals taken from populations collected in vineyards and citrus groves in Tavira, Portugal (37° 07′ N, 07° 39′ E). Both populations are characterized by absence of LI-attracted males. Crosses were also conducted between formerly laboratory-reared P. ficus females and Israeli P. ficus males that responded to LI obtained from a laboratory-reared population collected from vineyards in Lachish, Israel (31° 33′ 54″ N, 34° 50′ 56″ E). Mealybug populations were reared according to KolMaimon et al. (2014), with the following modifications: P. ficus were transferred as eggs to clean (mealybug free) potato sprouts 2 weeks earlier than P. citri, in order to synchronize the emergence of males with that of cross-breeding virgin females. The tested individuals of the Portuguese and Israeli P. ficus populations were reared coherently, i.e., male pupae and immature (L3) females were carefully removed from the rearing boxes, and those of each gender were placed in separate containers, in order to prevent uncontrolled copulations. When the females reached maturity (3–5 days after the last molt), newly emerged adult males were placed together
with the females for 4 days (the maximum adult male life span at temperatures under 25 °C) after pherotype characterization. Thus, P. citri males were exposed to P. ficus virgin females, and Israeli P. ficus males were exposed to Portuguese P. ficus virgin females. Each ovipositing female was transferred to fresh clean potato sprouts placed inside a container, for further observations. Male pherotype characterization In order to track male pherotypes, each of the tested males and their male offspring were characterized according to KolMaimon et al. (2014): Every male was exposed to three pheromones, each impregnated in a filter paper disk, and each lure was placed in a different arena. The pherotype of the tested male was determined according to its mode of attraction to each lure. In the P. ficus×P. citri cross-experiment, the tested pheromones were P. citri pheromone C and P. ficus pheromone LS; LI served as a control, because the males of neither population were attracted to LI. In the Portuguese × Israeli mealybug population cross-experiment, the tested pheromones were LS and LI, and C served as control. Induced hybridization For the first crossing of P. ficus females with P. citri males, three male pherotypes were used: (i) pherotype S—individuals attracted only to P. ficus pheromone LS; (ii) pherotype C—individuals attracted only to P. citri pheromone; and (iii) pherotype N—individuals attracted to neither of the tested pheromones. In the second crossing, Portuguese P. ficus females were mated with Israeli LI-attracted P. ficus males. In order to produce the second generation (F2) of the first cross, male pherotypes C, S, and N were used. In order to create F2 of the second cross, male pherotypes I, S, and N were used (see also Fig. 1). Male and female genotype characterization ITS2 gene sequencing was used for genotyping the parent populations—F1 and F2 hybrids of P. ficus females mated with P. citri males. ITS2 genotyping was monitored only for the cross between P. ficus and P. citri because no consistent differences between the two subpopulations of P. ficus were found. Segments of ITS2 gene were amplified, purified, and sequenced as recently described by Kol-Maimon et al. (2014). The CLUSTALW software (SFI, Dublin, Ireland) was used for multiple sequence alignment with GenBank reference sequences and for specimen genotyping. The following ITS2 GenBank references were used: P. ficus—GU134677, JQ085574, and HQ852471; P. citri—FJ430145 and JF714195.
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Fig. 1 A presentation of the crossing design. Two crosses are presented: (i) P. ficus [Pf] females mated with P. citri [Pc] males and (ii) Portuguese [PT] P. ficus females mated with Israeli [IL] P. ficus males. H refers to experimentally produced hybrid females or males, P parent generation, F1 first laboratory-crossed generation, F2 second laboratory-crossed and backcrossed generation. Signs inside a circle represent females, and signs
inside a square represent males. The last capital letter in males (in squares) represents the mode of pheromone male attraction (pherotype): N none attraction to neither of the tested pheromones, C attraction to P. citri pheromone alone, F attraction to P. ficus pheromone alone, I attraction to P. ficus pheromone LI alone, S attraction to P. ficus pheromone LS alone
Specimens with 98–100 % sequence identity with P. citri or P. ficus references were considered to be P. citri (type Pc) or P. ficus (type Pf), respectively. Specimens with less than 94 % identity with P. citri or P. ficus references were considered to be hybrids (type H). In order to demonstrate hybrid sequences (type H), further analysis of the chromatogram peak readings was conducted by converting peaks to probabilities of nucleotides occurring in each position (see Tables S1 and S2). This analysis was conducted with the MUSCLE alignment software (Edgar 2004) and BioPerl (ActiveState Software Inc., Vancouver, BCV6C 1 T2, Canada) for nucleotide probabilities per position.
(ii) P. ficus, and for the Portuguese × Israeli cross, the two groups were (i) hybrids and (ii) Portuguese population.
Statistical analysis Pearson contingency analysis was conducted for comparison of pherotype and genotype distributions between generations, i.e., comparisons among distributions of parent populations, F1 hybrids, and F2 hybrids, by using the JMP software version 10 (SAS institute Inc., Cary, NC, USA). In order to simplify the analysis and minimize the number of compared groups which did not differ significantly, all male pherotypes used to produce F2 progeny were grouped together. Thus, only two groups of males were used for comparing the frequencies in the next generation and the backcrosses. For the P. ficus×P. citri cross, the two groups were (i) hybrids and
Results Pherotype distribution among hybrids of P. ficus females mated with P. citri males The pherotypes C (attracted only to P. citri pheromone) and FC (attracted to pheromones of both species) were present in the paternal P. citri population (23 % for C and 39 % for FC) but absent from the maternal P. ficus population (Fig. 2). The pherotypes F (males attracted only to P. ficus pheromone) and N (males attracted to neither of the tested pheromones) were also detected in parental populations: 66 and 34 %, respectively, of the sampled P. ficus males and 11 and 27 %, respectively, of the P. citri males (Fig. 2). Among F1 male hybrids, 50, 44, 4, and 3 % were F, N, C, and FC pherotypes, respectively (Fig. 2). Contingency analysis showed a significant difference between pherotype distributions of the parent generation and of the F1 hybrid generation (P. ficus vs. F1 P
