Application of microsatellite primers for the social wasp Polistes to another social wasp genus, Parapolybia, to estimate genetic relationships among nestmates
Abstract
We tested 14 pairs of microsatellite primers that had been developed for the genus Polistes to assess their applicability for determining genetic structure in a colony of the genus Parapolybia. At least one of the 14 Polistes primer sets was useful for Parapolybia indica, and the locus identified by this primer set was highly polymorphic. Using this primer set, we estimated the average relatedness among female nestmates of Parapolybia indica to be 0.82 ± 0.05 (mean ± SE), indicating that in this species single mating is normal, although some cases of multiple mating was found.
INTRODUCTION
Microsatellite genetic loci comprise simple sequence repeat regions, and are very useful for the study of social structures and social interactions, and consequently the evolution of altruistic behavior in social insects (Queller et al. 1993; Hughes 1998). These repeat regions are easily amplified by polymerase chain reaction (PCR), can be highly polymorphic, and the resulting data can be easily interpreted. Using polymorphic markers it is possible to estimate relatedness, effective queen number, mating frequency, and degree of inbreeding, and to identify diploid males and characterize the genetic structure of colonies and/or populations. Unfortunately, carrying out searches for useful loci is both costly and time consuming. However, in recent years, many microsatellite primers have been developed for various animals and plants and the sequences of these primers have been deposited in data banks such as GenBank. Microsatellite primers developed for a given species are sometimes useful for other species, in both the same genus and other genera within the same family (Ezenwa et al. 1998; Henshaw et al. 2001), so it is possible to search for pre-existing suitable primers in the data banks, which is the approach we took.
The social wasp genus Parapolybia (Hymenoptera, Vespidae, Polistinae, Ropalidiini) consists of five known species, and is distributed from Iran in the west, to Japan, the Philippines, and New Guinea in the east (Das & Gupta 1989; Gadagkar 1991). Few studies have been carried out on the ecology and behavior of this small group (Sekijima et al. 1980; Kojima 1983; Sugiura et al. 1983a,b; Yamane 1985; Sasai & Matsuura 1986; Suzuki 2003; see also Gadagkar 1991) in comparison with other related social wasps, for example Polistes and Ropalidia. There are two species of Parapolybia in Japan, P. indica (de Saussure) and P. varia (Fabricius). The two species are likely to have different social lives in temperate Japan and in the tropics. This enables us to compare the different life histories and genetic structures at the colony and population level in different environmental conditions. For such studies, genetic analyses are needed. To date, there have been no genetic studies of Parapolybia, because no specific microsatellite primers are available. In the present study, we examined whether existing primers developed for Polistes could be used for Parapolybia. We then used one primer set to study the genetic relationships among P. indica nestmates.
MATERIALS AND METHODS
Sample specimens
We used both Parapolybia indica and P. varia in the search for useful microsatellite loci. Parapolybia indica was collected in Fukushima Prefecture and Tokyo, Japan, and P. varia was collected in Nakanoshima, Kagoshima Prefecture, Japan, and Puri, Taiwan (two specimens provided by J. Takahashi). For analyzing the genetic relationships among nestmates, P. indica colonies were collected in Kanagawa and Fukushima Prefectures, Japan. All insect samples were kept in 99.5% ethanol until DNA extraction.
Search for useful microsatellite loci
We tested 14 microsatellite primers from Polistes (Table 1). Genomic DNA of Parapolybia wasps was extracted from the hind leg muscle using a proteinase K phenol-chloroform method modified from the method of Sambrook et al. (1989). PCR amplification of microsatellite DNAs was carried out using a programmable thermal cycler (PTC-100; MJ Research, Waltham, MA, USA) in 10 µL of reaction mixture containing 10–100 ng of template DNA, 0.2 mM dNTPs, 0.2 pM of each primer, ddH2O, 1× Gene Taq universal buffer (Nippon Gene, Tokyo, Japan; including 1.5 mM Mg2+), and 0.5 U Gene Taq NT polymerase (Nippon Gene). Thirty PCR cycles of 20 s at 94°C, 1 min at 50°C, and 2 min at 72°C were performed following an initial denaturation step at 94°C for 2 min. The PCR products were electrophoresed on 300 V (constant voltage) for 1.5 h in 6% denaturing (36% urea) polyacrylamide gels or non-urea 8% polyacrylamide gels (1× TBE (0.09 M Tris, 0.09 M boric acid, 0.002 M ethylenediamine tetraacetic acid), 200 mm × 200 mm × 1 mm). The genotype of each individual was detected by using the silver-stain method (Tegelström 1986).
| Locus name | GenBank accession numbers | Reference | P. indica | P. varia | Product size (bp) | Availability | ||
|---|---|---|---|---|---|---|---|---|
| n | No. bands | n | No. bands | |||||
| PBE216 | U64623 | S | 4 | 1 | 4 | 2–3 | <100 | Available? |
| PBE269 | U64624 | S | 4 | MB | 4 | MB | – | No |
| PBE411 | U64625 | S | 4 | 2–3 | 4 | 2–3 | 150–170 | Available? |
| PBE442 | U64632 | S | 4 | NP | 4 | NP | – | No |
| PBE492 | U64635 | S | 4 | NP | 4 | NP | – | No |
| Pc01 | AB083012 | T | 20 | NP | – | – | – | No |
| Pc14 | AB083015 | T | 20 | MB | – | – | – | No |
| Pc17 | AB083016 | T | 20 | MB | – | – | – | No |
| Pc27 | AB083017 | T | 20 | NP | – | – | – | No |
| Pc34 | AB083018 | T | 20 | MB | – | – | – | Available? |
| Pc41 | AB083019 | T | 20 | NP | – | – | – | No |
| Pc63 | AB083021 | T | 20 | 1–2 | – | – | 130–180 | Available |
| Pc68 | AB083022 | T | 20 | MB | – | – | – | Available? |
| Pc80 | AB083023 | T | 20 | 1 | – | – | – | No |
- MB, multiple scattered bands; n, sample size; NP, no scorable product because of smeared bands; S, Strassmann et al. (1997); T, Tsuchida et al. (2003).
Genetic structure in the Parapolybia indica colony
A total of eight foundress queens, 214 adult females (presumably the foundress queens’ daughters) and 88 adult males from the 25 colonies collected in Fukushima and Kanagawa Prefectures were analyzed using the most appropriate microsatellite locus. DNA extraction, PCR, electrophoresis (non-urea) and silver-staining were carried out as described above. Genetic relatedness was calculated using Relatedness 4.2c software (Queller & Goodnight 1989; Goodnight & Queller 1994).
RESULTS
Bands were visible for the PCR products amplified using nine of the PCR primer sets (Table 1). Of these nine primer sets, one (Pc63) yielded one or two clear bands for P. indica, depending on the individual, at a position corresponding to one locus, indicating the presence of a polymorphic microsatellite locus (Pc63, F: 5′-gac cct aat tct ctt gct gg-3′, R: 5′-gtg atc gtg tcg cgt gtt aa-3′). Primers for other loci yielded only a single band (indicating that the locus was monomorphic), a few bands (probably indicating insufficient resolution, although possibly also indicating the presence of a polymorphic locus), or smeared bands (uncountable) or multiple scattered bands, which were unsuitable as genetic markers.
We then used the locus Pc63 to determine the genetic relatedness of nestmates in the P. indica colonies. Of the DNA samples from 310 individuals, samples from 303 were amplified successfully (97.7%). The genotypes of eight foundress queens, 211 adult females and 84 adult males from 25 colonies in four subpopulations were examined (Table 2).
| Colony code | Location | Foundress queen present | No. females | No. males | Collection date |
|---|---|---|---|---|---|
| 010704-1 | K-Y | No | 15 | 0 | 29/7/2001 |
| 010704-3 | K-O | No | 5 | 0 | 20/7/2001 |
| 010708-1 | K-Y | No | 12 | 0 | 26/7/2001 |
| 010708-2 | K-Y | No | 12 | 1 | 26/7/2001 |
| 010712-1 | K-H | No | 4 | 0 | 25/7/2001 |
| 010712-2 | K-H | Yes | 0 | 19 | 25/7/2001 |
| 010712-3 | K-H | No | 4 | 0 | 2/8/2001 |
| 010712-5 | K-H | No | 4 | 0 | 25/7/2001 |
| 010712-6 | K-H | Yes | 0 | 20 | 22/7/2001 |
| 010715-3† | K-H | Yes | 0 | 0 | 17/8/2001 |
| 010715-4 | K-H | No | 6 | 0 | 23/8/2001 |
| 010715-5 | K-H | No | 4 | 0 | 17/8/2001 |
| 010715-7 | K-H | No | 4 | 0 | 25/7/2001 |
| 010718-1 | K-Y | No | 11 | 2 | 18/7/2001 |
| 010718-2b | K-Y | No | 6 | 0 | 26/7/2001 |
| 010718-3 | K-Y | Yes | 3 | 0 | 26/7/2001 |
| 010720-1† | K-O | Yes | 0 | 0 | 29/7/2001 |
| 010720-3 | K-O | Yes | 10 | 29 | 26/7/2001 |
| 010726-1 | K-Y | No | 14 | 17 | 11/8/2001 |
| 010726-2 | K-Y | No | 9 | 0 | 26/7/2001 |
| 010729-4 | K-O | Yes | 10 | 0 | 29/7/2001 |
| 010806-1 | F-A | Yes | 35 | 0 | 9/8/2001 |
| 010806-2 | F-A | No | 22 | 0 | 9/8/2001 |
| 010813-1 | K-O | No | 14 | 0 | 13/8/2001 |
| 010817-3 | K-H | No | 10 | 0 | 17/8/2001 |
| Total | 4 sites | 8 present | 214 | 88 |
- Females with shabby forewings were regarded as foundress queens, and “females” refers to female nestmates excluding the foundress queen. †Only the foundress queen was collected. K-Y, K-O, K-H: Yokosuka, Ohiso, and Hadano, respectively, Kanagawa Prefecture; F-A, Aizu-Tajima, Fukushima Prefecture.
The microsatellite locus Pc63 carried nine alleles, which we named A to I (Tables 3,4). As expected, the diploid females yielded one or two bands, whereas the haploid males yielded only one band.
| Colony | Foundress queen Genotype | Female genotype† | Male genotype | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | B | C | D | E | F | G | H | I | [Failure] | |||
| 010704-1 | GH (15) | |||||||||||
| 010704-3 | HH (4), HI (1) | |||||||||||
| 010708-1 | FG (3), FI (9) | |||||||||||
| 010708-2 | BF (1), BG (11) | 1 | ||||||||||
| 010712-1 | AF (1), FH (3) | |||||||||||
| 010712-2 | AH | – | 10 | 7 | 2 | |||||||
| 010712-3 | AA (2), AH (2) | |||||||||||
| 010712-5 | CH (4) | |||||||||||
| 010712-6 | EH | – | 9 | 11 | 0 | |||||||
| 010715-3 | [failure] | – | ||||||||||
| 010715-4 | BH (2), BI (4) | |||||||||||
| 010715-5 | BB (1), BH (3) | |||||||||||
| 010715-7 | EG (4) | |||||||||||
| 010718-1 | GH (11) | 2 | 0 | |||||||||
| 010718-2b | DH (5), DI (1) | |||||||||||
| 010718-3 | GH | GH (2), HH (1) | ||||||||||
| 010720-1 | GG | – | ||||||||||
| 010720-3 | AH | AG (2), GH (7), [failure, 1] | 15 | 14 | 0 | |||||||
| 010726-1 | CC (1), CH (2), HH (10) | 15 | 2 | |||||||||
| [failure, 1] | ||||||||||||
| 010726-2 | II (9) | |||||||||||
| 010729-4 | GH | EG (4), EH (5), GH (1) | ||||||||||
| 010806-1 | BG | BB (23), BG (10), HH (1), [failure,1] | ||||||||||
| 010806-2 | AG (1), AH (6), HH (15) | |||||||||||
| 010813-1 | GG (14) | |||||||||||
| 010817-3 | BE (5), BI (5) | |||||||||||
- Capital letters indicate the genotypes of each individual. †The number of females is given in parentheses. “Failure” indicates that it was not possible to genotype an individual.
| Deme | No. colonies | No. females | Allele frequency | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| A | B | C | D | E | F | G | H | I | |||
| Ohiso† | 4 | 38 | 0.037 | 0.0 | 0.0 | 0.0 | 0.150 | 0.0 | 0.583 | 0.230 | 0.0 |
| Hadano† | 7 | 36 | 0.125 | 0.232 | 0.071 | 0.0 | 0.107 | 0.071 | 0.071 | 0.238 | 0.083 |
| Yokosuka† | 8 | 80 | 0.0 | 0.056 | 0.009 | 0.056 | 0.0 | 0.060 | 0.213 | 0.433 | 0.173 |
| Aizu Tajima‡ | 2 | 55 | 0.071 | 0.412 | 0.0 | 0.0 | 0.0 | 0.0 | 0.074 | 0.433 | 0.0 |
| Total | 21 | 209 | 0.054 | 0.140 | 0.028 | 0.024 | 0.057 | 0.050 | 0.205 | 0.340 | 0.102 |
- † Kanagawa Prefecture.
- ‡ ‡ Fukushima Prefecture.
Foundress queens, females and males (where present) were genotyped in six colonies (Table 3). In these queenright colonies: (i) males were the foundress queen’s sons, judging from the genotypes of both (colonies 010712-2, -6, 010720-3), although females were absent at the time of collection in these colonies; (ii) single mating occurred in colonies 010718-3 and 010720-3, and multiple mating occurred (at least twice) in colony 010729-4, if females did not drift between colonies; (iii) at times, females may have drifted between colonies, given the presence of females that were genetically unrelated to the foundress queen (010806-1). It is possible that either multiple mating or drifting occurred in colony 010806-2.
The gene frequencies for females (209 females from 21 colonies from four localities) were calculated (Table 4). Allele H dominated, and alleles B or G were next most common, depending on the locality. We estimated the average relatedness among female nestmates to be 0.82 ± 0.05 (mean ± SE; excluding two females in colonies 010806-1 and 010806-2 that had presumably drifted there). Although multiple mating might have occurred in some colonies, these values did not differ significantly from 0.75, which represents the relatedness between full sisters in a haplo-diploid species (t-test, ts = 1.49, d.f. = 20, P > 0.1).
DISCUSSION
Henshaw et al. (2001) recently identified 52 microsatellite loci in Polybioides tabidus (Fabricius) and used primers for these loci to amplify DNA from 11 species of eusocial wasps, including a single specimen of Parapolybia varia. Their study showed that some primers might be useful for P. varia, with some loci apparently being polymorphic.
In the present study, we found that at least one primer developed for Polistes chinensis antennalis (Pérez) was useful for identifying a polymorphic microsatellite locus in P. indica. In this study, we fixed the PCR annealing temperature at 50°C to simplify our procedure. If different conditions were to be used for PCR, some of the other primers might also be found to be useful.
Genetic analyses have shown that in eusocial wasps, such as the Polistinae, mating usually occurs once (Strassmann et al. 1989; Tsuchida 1994; Sayama & Takahashi 2005). In the present study the average relatedness among P. indica sisters was very high (0.82 ± 0.05), and did not differ significantly from 0.75, which suggests that each foundress queen mates only once and is the mother of all female offspring. We did, however, find some multiple matings, but further study is needed to clarify how frequently multiple matings occur.
ACKNOWLEDGMENTS
We are grateful to Dr T. Kusano, Dr F. Hayashi, and Dr S. J. Martin for their invaluable comments on the manuscript. We also thank Dr J. Takahashi for advice on primer selection. This study was supported in part by a Grant-in-Aid for Basic Research from the Japan Ministry of Education, Science and Culture (no. 12304048).
