Introduction

Inherited symbionts that distort host reproduction to their own benefit are common in insects. One strategy is that of male-killing, in which male hosts are killed by the symbiont to the benefit of females infected with the same symbiont strain. Aphidophagous coccinellid beetles that lay eggs in clutches and in which neonate larvae indulge in sibling egg consumption have been identified as a group that is particularly prone to invasion by male-killers (Majerus & Hurst, 1997). To date, maternally inherited female-biased sex ratios have been recorded in nine species of coccinellid (Lusis, 1947; Shull, 1948; Matsuka et al., 1975; Niijima & Nakajima, 1981; Niijima, 1983; Hurst et al., 1992; 1997a; Majerus & Hurst, 1997; Majerus et al., 1998; 1999a; Majerus & Majerus, in press). These sex ratio biases have been shown or are assumed to result from male-killing symbionts, with a diverse array of bacteria being associated with the male-killing trait. In Adalia bipunctata, at least four different bacteria are known to cause male-killing: a Rickettsia (Werren et al., 1994a), Spiroplasma (Hurst et al., 1999b) and two strains of Wolbachia (Hurst et al., 1999c).

Theoretical models of male-killers, based on vertical transmission efficiency, cost of infection borne by infected females and the level of fitness compensation (the benefit that accrues to surviving offspring of infected females as a result of the death of males), suggest that two or more male-killers cannot stably coexist in a single population (Hurst, 1991; Hurst et al., 1997b). As Randerson et al. (2000) have shown, the male-killer with the highest basic rate of increase (BRI), which is a product of the male-killer’s vertical transmission efficiency, its direct costs on female bearers and the level of fitness compensation, will out-compete all others.

Despite this theoretical prediction, in several locations different male-killers reported from A. bipunctata are known to coexist. Thus, in Berlin, both the Rickettsia and the Spiroplasma coexist (Hurst et al., 1999b), while in Moscow, the Spiroplasma coexists with two strains of Wolbachia (Hurst et al., 1999c). Speculative explanation of such coexistence has hitherto considered hybrid zones between regions of pure infection, possibility of a transient situation consequent on recent secondary invasion and the possibility of host genes that affect the transmission efficiency of the male-killers (Hurst et al., 1999c). We here report analysis of male-killing in a single sample of A. bipunctata showing the coexistence of four different male-killers in this species and discuss the tenability of the theoretical explanations of male-killer coexistence.

Materials and methods

Phenotypic assaying of male-killing

A sample of 110 A. bipunctata was collected on 11 October 1997, from overwintering aggregations in a building near the Vavilov Institute of General Genetics, Gubkin Street, Moscow. The sample was transported to Cambridge in January 1998, where it was retained at 4°C until April. Approximately 100 ladybirds were then removed from the refrigerator, placed in clean stock Petri-dishes, fed initially on an artificial food (Majerus et al., 1989) for 2 days and thereafter on pea aphids, Acyrthosiphon pisum (Harris). Mating pairs where removed to individual Petri-dishes as they formed. In this way 40 mating pairs were isolated. These pairs were initially assayed for male-killer presence using two routine phenotypic indicators: egg hatch rates and progenic sex ratio (Hurst et al., 1992). In short, eggs were collected and counted from pairs daily, egg hatch rates were recorded, and resulting adults were sexed under CO2 anaesthetic.

F1 crosses, using at least three females from each of the lines which produced a significantly female-biased sex ratio and males from normal lines, were set up to show that the female bias was inherited. Again egg hatch rates and progenic sex ratios were recorded. Vertical transmission rates for the different male-killers were assessed using progenic sex ratios from F1 and F2 crosses of matrilines shown to bear a male-killer.

Identification of male-killing agents

The respective symbionts were characterized using PCR assays specific for the known male-killing bacteria of A. bipunctata as described in Hurst et al. (1999c). Females with both low egg hatch rates and significantly female-biased progenic sex ratios were tested for each of the above-identified male-killing lineages. Genomic DNA was isolated from ovaries and subsequently subjected to PCR. For PCR tests, we used primers specific for the citrate synthase gene of male-killing Rickettsia (RICS741F: 5′-CATCCGGAGCTAATCCTTTTGC; RCIT1197R: 5′-CATTTCTTTCCATTGTGCCATC; cf. Davis et al., 1998), and for the ribosomal spacer region of Spiroplasmas of the S. ixodetis group (SPITS-J04: 5′-GCCAGAAGTCAGTGTCCTAACCG; SPITS-N55: 5′-ATTCCAAGGCATCCACCATACG; cf. Schulenburg et al. submitted). For identification of Wolbachia, we used the previously published primers for the wsp gene (primers wsp81F and wsp691R; Zhou et al., 1998). PCR suitability of genomic DNA preparations was checked via amplification of the mitochondrial cytochrome oxidase subunit I gene using insect-specific primers (Simon et al., 1994). Identities of Wolbachia strains were established by DNA sequencing. Amplified wsp sequences were cloned and subsequently sequenced for both strands. Three clones per host specimen were analysed to guard against PCR errors. The generated sequences were thereafter compared to those of the two male-killing Wolbachia strains ‘Y’ and ‘Z’ which were previously isolated from A. bipunctata (formerly strains ‘A’ and ‘B’, respectively; now renamed in order to avoid confusion with nomenclature for the Wolbachia subgroups as both belong to same subgroup B). For details of molecular methods, see Hurst et al. (1999c).

Results

Phenotypic assay for the male-killing trait

Four of the 40 initial pairs failed to produce sufficient progeny for analysis. Of the remainder, six produced both low egg hatch rates (<0.55) and significantly female-biased sex ratios (Table 1). All except two of the F1 families using females from these lines (n=25) produced significantly female-biased sex ratios. The exceptions were one female of four from Mos 3, and one female of five from Mos 18, both of which produced high egg hatch rates and normal sex ratios.

Table 1 Egg hatch rates and progenic sex ratios of lines of Adalia bipunctata showing the male-killing trait
Full size table

Identity of male-killing agents

Female progeny from each of the six significantly female-biased matrilines amplified with one, and only one, of the primer pairs used. One line (Mos 3) amplified with the Rickettsia primers, three (Mos 9, Mos 33 and Mos 35) with the Spiroplasma primers, and two (Mos 6 and Mos 18) with the Wolbachia primers. DNA sequences of the wsp gene showed the Wolbachia from the two host lineages to be different. The wsp sequence isolated from line Mos 6 is identical to the strain “Z” Wolbachia of A. bipunctata, whereas that for Mos 18 is identical to that of strain ‘Y’.

Vertical transmission efficiencies

With the exclusion of the two revertant F1 families, the vertical transmission rates, calculated as 1 − (the number of males produced divided by the number of females produced) (Hurst et al., 1993), were consistent between generations for individual matrilines, but varied considerably between matrilines (Table 2). There was significant heterogeneity between the sex ratios resulting from the four male-killers (χ23=106.588, P < 0.001 (summing the three Spiroplasma matrilines)), indicating variation in vertical transmission rates of the male-killers. Of further note, the vertical transmission efficiencies of the two strains of Wolbachia differed significantly (χ21=17.563, P < 0.001), the ‘Z’ strain giving rise to proportionately more males than the ‘Y’ strain.

Table 2 Vertical transmission efficiencies from six matrilines bearing male-killing bacteria
Full size table

Discussion

Consideration of the known geographical distributions of the four male-killer strains found in this single Muscovite sample make it highly unlikely that their coexistence represents a ‘hybrid zone’. The A. bipunctata Rickettsia male-killer has been recorded from England, Germany and Denmark in Europe (Hurst et al., 1992; 1999b) and Kirgisia in Asia (Zakharov et al., 1998). Male-killing Spiroplasmas have been recorded in A. bipunctata from Central and Eastern European localities (Bielefeld, Bayreuth, Berlin, St. Petersburg, Moscow) (Hurst et al., 1999b), and from Asia (Tuva; Schulenburg et al. submitted). All of them have identical ribosomal spacer sequences (Schulenburg et al. submitted). The Rickettsia and Spiroplasma male-killers thus have wide geographical distributions which seem to overlap from Central Europe into Asian Russia. The two male-killing Wolbachia strains have been recorded from Moscow (Hurst et al., 1999c) and Tomsk (Zakharov and Schulenburg, pers. obs.).

A second hypothesis for coexistence is that the Muscovite population of A. bipunctata is heavily substructured and not panmictic as the models assume. For example, one might imagine a ‘haystack model’ in which local colonies exist with little migration between them. In such a situation, different male-killers may exist in matrilines adapted to different environmental conditions (e.g. different host plants). Studies of dispersion of A. bipunctata throughout the year argue strongly against heavy substructuring of populations. The typical pattern is of a dispersal flight in the spring, from overwintering sites away from host plants (usually in or on buildings). Initial activity in spring involves feeding on alternative foods and frequent flights in search of suitable essential prey (sensu Hodek, 1973). Once essential food has been found, mating and oviposition take place, females mating with many males during their reproductive life. Females move frequently during their reproductive period, both between patches of one type of host plant and different species of host plant, dispersal to new sites being initiated by the presence of coccinellid larvae (e.g. Hemptinne et al., 1992). Males follow females after a lag of a few days (Brakefield, 1984). At the end of the reproductive period, adults of one or more generations produced during the year disperse away from host plants to find suitable places to overwinter, often forming large overwintering aggregations (Majerus, 1994). The whole pattern is of a highly mobile well mixed population. Movement during the spring and summer is driven by the need to find suitable colonies of aphids, for which their is strong competition, both within the species, and with other predators and parasitoids. The differences in the pattern of usage of aphids on different species of host plants between different years (e.g. Majerus & Kearns, 1989) argues against females being adapted to particular host plants.

A third hypothesis, that the coexistence of different male-killers results from very recent novel invasions of some of the male-killers into A. bipunctata, also seems untenable. In the case of the Rickettsia and the Spiroplasma, again the wide area of geographical overlap argues against either being of recent occurrence. For the two male-killing strains of Wolbachia from A. bipunctata, phylogenetic analysis of wsp sequences suggests that the two have a common origin but did not diverge recently (Hurst et al., 1999c). The most likely explanation is of a single, not recent origin of male-killing Wolbachia in A. bipunctata.

A fourth hypothesis, that host suppresser genes exist which differentially affect the transmission of the various male-killers is plausible and would help maintain the polymorphism (Randerson et al., 2000). However, an analysis of whether suppresser genes could maintain as many as four different male-killers within the same population seems inappropriate until evidence of the existence of such genes in coccinellids has been obtained.

None of these explanations of the coexistence of the four male-killers (and there may be more) in Muscovite A. bipunctata currently appears tenable. The unexpectedness of the coexistence of these four male-killers within a single population is consequent both upon models of male-killer population dynamics, and on the competitive exclusion principle.

To address the dichotomy between the observations of multiple male-killers contained herein and predictions made by models of the evolutionary dynamics of male-killing endosymbionts, future work should seek to: (i) verify the assumptions of the models; (ii) test the applicability of laboratory-generated data on vertical transmission efficiencies and direct costs on female hosts to field situations; (iii) determine whether there is any niche separation in the utilization of hosts by symbionts; (iv) monitor fluctuations in the prevalences of the four symbionts over time; and (v) determine whether all A. bipunctata individuals are equally susceptible to each of the male-killers.

Until potential variability in the interactions between the different male-killers and their hosts have been investigated, it is premature to view this as a case where the competitive exclusion principal is being compromised.

A detailed, field based, comparative analysis of the vertical transmission efficiency of the different male-killers, the occurrence of horizontal transmission, and the fitness benefits that accrue to surviving larvae of male-killed clutches for each symbiont, will be needed to assess levels of niche separation between these symbionts and solve the conundrum of the presence of multiple male-killer agents in A. bipunctata.