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Proceedings of the Royal Society B: Biological Sciences logoLink to Proceedings of the Royal Society B: Biological Sciences
. 2018 Apr 18;285(1877):20180369. doi: 10.1098/rspb.2018.0369

Rapid comeback of males: evolution of male-killer suppression in a green lacewing population

Masayuki Hayashi 1,2, Masashi Nomura 2, Daisuke Kageyama 3,✉
PMCID: PMC5936734  PMID: 29669904

Abstract

Evolutionary theory predicts that the spread of cytoplasmic sex ratio distorters leads to the evolution of host nuclear suppressors, although there are extremely few empirical observations of this phenomenon. Here, we demonstrate that a nuclear suppressor of a cytoplasmic male killer has spread rapidly in a population of the green lacewing Mallada desjardinsi. An M. desjardinsi population, which was strongly female-biased in 2011 because of a high prevalence of the male-killing Spiroplasma endosymbiont, had a sex ratio near parity in 2016, despite a consistent Spiroplasma prevalence. Most of the offspring derived from individuals collected in 2016 had 1 : 1 sex ratios in subsequent generations. Contrastingly, all-female or female-biased broods appeared frequently from crossings of these female offspring with males derived from a laboratory line founded by individuals collected in 2011. These results suggest near-fixation of a nuclear suppressor against male killing in 2016 and reject the notion that a non-male-killing Spiroplasma variant has spread in the population. Consistently, no significant difference was detected in mitochondrial haplotype variation between 2011 and 2016. These findings, and earlier findings in the butterfly Hypolimnas bolina in Samoa, suggest that these quick events of male recovery occur more commonly than is generally appreciated.

Keywords: endosymbiont, evolutionary arms race, genetic suppression, green lacewing, sex ratio bias, Spiroplasma

1. Introduction

Selfish genetic elements are characterized by their ability to spread in a population despite the harm they impose on their hosts [1–5]. Cytoplasmic elements (e.g. maternally transmitted endosymbionts and organelles) are not transferred to the next generation through sperm and hence can be advantageous if they distort the sex ratio towards females. Nuclear genes, however, can be selectively favoured if they can return the population to a 1 : 1 sex ratio: there is a so-called cytonuclear conflict or evolutionary arms race over control of the sex ratio [6–8]. Some arthropod species are infected with maternally transmitted endosymbionts that distort the host sex ratio towards females by killing or feminizing males [9,10]. The existence of host suppressors acting against cytoplasmic sex ratio distorters has been reported in several species of arthropods such as lady beetles [11], Drosophila fruit flies [12], woodlice [13] and dwarf spiders [14]. However, the spread of a suppressor in a host population has been described only in a single species, the butterfly Hypolimnas bolina [15–18].

In a Samoan island population of H. bolina, an extremely female-biased sex ratio (about 100 females per male) was first reported in the early twentieth century [19,20]. In 2000, a similarly skewed sex ratio in this population was discovered, and the cause of the skew was revealed to be the prevalence of a cytoplasmically inherited Wolbachia endosymbiont [21,22]. Subsequent examinations of the population in 2005 and 2006 revealed transition of the population sex ratio to nearly 1 : 1, despite the near fixation of Wolbachia [16]. Cross-breeding experiments revealed that the 1 : 1 sex ratio was owing to the presence of a dominant zygotic suppressor against male killing [17]. It has been estimated that the suppressor spread very rapidly in the H. bolina population until it reached near fixation (within about 10 generations) [16].

Here, we show the dynamics of interaction between the green lacewing Mallada desjardinsi (Neuroptera: Chrysopidae) and its endosymbiont Spiroplasma in a natural population. In 2011, we found that 73.5% of M. desjardinsi females in a population in Matsudo, in central mainland Japan, were infected with Spiroplasma, which usually kills all male offspring during the embryonic and larval stages, resulting in a strongly female-biased sex ratio (57 females to seven males) [23]. Under this high frequency of male killing and the consequent female bias in the population sex ratio, nuclear suppressors against male killing were expected to spread in the population [18]. Five years after the survey, we therefore reinvestigated the sex ratio and the rate of Spiroplasma infection in the same population, and performed cross-breeding experiments to look for the presence of host nuclear suppressors of male killing.

2. Material and methods

(a). Collection and culture of lacewings

Green lacewings M. desjardinsi were collected and bred in a manner similar to that described previously [23]. We caught a total of 129 adults with an insect net on the campus of Chiba University, Matsudo, Chiba, Japan, at night (from 20.00 to 22.00) from June to November 2016. Collected individuals were sexed by their abdominal tip morphology. Some females (n = 33) were individually allowed to lay eggs in plastic cases (120 mm diameter, 100 mm height) for 7 days while being provided an artificial diet (50% honey solution and a paste of dried yeast). All eggs laid on the inside wall and lid were collected every 3 days and individually placed into a single well of a 24-well plate (cat. no. 142 475, Nunc Cell-Culture Treated Multidishes, Thermo Fisher Scientific K.K., Yokohama, Japan) together with a spoonful of eggs of the flour moth Ephestia kuehniella (Agrisect Inc., Ibaraki, Japan) as a larval food source. After egg collection, M. desjardinsi females were stored at −40°C until DNA extraction. Offspring were sexed after adult eclosion. The lacewings were reared in a climate-controlled room (25 ± 2°C; light : dark regime of 16 : 8 h).

The sex ratio of each brood was tested by an exact binomial test (EBT) to detect the bias from 1 : 1. In each brood in 2011 and 2016, survival rate data were analysed by a generalized linear model (GLM) with a binomial error distribution and logit link function. A model was constructed by using survival rate (i.e. number of adults versus number of dead before adult eclosion) as a response variable, and the infection status of mothers (i.e. positive and negative for Spiroplasma) as a fixed effect. The effect of Spiroplasma infection was tested by using the likelihood-ratio χ2 (LR) test. Statistical analyses were conducted with R v. 3.3.2 software [24].

(b). Cross-breeding

To reveal the presence of host nuclear suppressors, we crossed daughters of wild-caught M. desjardinsi females with males from a laboratory line (the ‘2011 line'; figure 1a) founded by females collected at the same site (the campus of Chiba University) in 2011 and maintained since. The 2011 line was confirmed to be Spiroplasma-free by polymerase chain reaction (PCR) assay. Because 21 out of 25 females that were infected with Spiroplasma produced only females in 2011 (electronic supplementary material, table S1) [23], we assumed that the frequency of individuals possessing suppressors—if indeed they existed—against male killing was low in the 2011 line. We therefore predicted that backcrossing the Spiroplasma-infected 2016 females with 2011 males, which involve partial genomic replacement, could allow Spiroplasma to express male killing.

Figure 1.

Figure 1.

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Sex ratios of offspring produced by Mallada desjardinsi females collected in (a) 2011 and (b) 2016. Spiro+, broods produced by Spiroplasma-infected females; spiro−, broods produced by uninfected females. ‘Female-biased' indicates male-containing broods with sex ratios significantly deviated from 1 : 1 (p < 0.05 by EBT); ‘normal' indicates broods with sex ratios not significantly deviated from 1 : 1 (p > 0.05 by EBT). Numbers of males and females of each brood are given in the electronic supplementary material, table S1. Data for 2011 are from [23].

Nine daughters produced by three Spiroplasma-positive females (no. 6, no. 9 and no. 11) collected in 2016 (see Results and electronic supplementary material, table S1) were individually coupled with males of the 2011 line and allowed to lay eggs in plastic cases as described above (outbred scheme, figure 2a). As a control, 10 daughters of the same brood were coupled with sons produced by the wild-caught females (no. 6, no. 9 or no. 11) (inbred scheme, figure 2a). Note that the terms ‘outbred' and ‘inbred' are used simply for convenience to represent whether mated males were derived from the 2011 line or the 2016 line; they do not strictly reflect the genetic status of the matings. Eggs laid by the daughters were reared and the adults were sexed as above. After oviposition, the Spiroplasma infection status of each mother was diagnosed by PCR.

Figure 2.

Figure 2.

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Crossing scheme of breeding experiments (a) and sex ratios (proportions of male offspring) of (b) F1 and (c) F2 offspring derived from three wild-caught Spiroplasma-infected females of Mallada desjardinsi (no. 6, no. 9 and no. 11) that were crossed with 2011 males (outbred) and 2016 males (inbred). Box plots show medians, quartiles, ranges and outliers. Numbers of males and females in each brood are given in the electronic supplementary material, table S2. (Online version in colour.)

The sex ratio data from the crossing experiments were analysed by using a generalized linear mixed model with a binomial error distribution and a logit link function. A model was constructed using sex ratio (i.e. number of males versus number of females) as a response variable, the male line (i.e. 2011 and 2016) as a fixed effect, and the brood nested within a matriline of the female (i.e. no. 6, no. 9 and no. 11) as a random effect. The effect of a male line was tested by the LR test. These analyses were conducted with the lme4 package in R software.

For some lines, we continued to cross in the second and third generations. The sex ratios of brood and infection status of mothers were determined by PCR.

(c). Molecular identification of Spiroplasma

DNA was extracted from the abdomens of adult M. desjardinsi by using a DNeasy Blood and Tissue Kit (Qiagen K.K., Tokyo, Japan). A partial sequence of spoT, a Spiroplasma gene sequence similar to that of the (p)ppGpp 39-pyrophosphohydrolase spoT gene [25], was PCR amplified by using a pair of primers, spoT-f (5′-CAAACAAAAGGACAAATTGAAG-3′) and spoT-r (5′-CACTGAAGCGTTTAAATGAC-3′) [23,26]. The PCR products were subjected to ExoSAP-IT product clean-up (Thermo Fisher Scientific K.K., Yokohama, Japan) and then directly sequenced by Eurofins Genomics K.K. (Tokyo, Japan).

(d). Mitochondrial haplotypes

If the Spiroplasma was replaced with a non-male-killing variant between 2011 and 2016, a mitochondrial haplotype associated with the Spiroplasma variant was expected to spread in the lacewing population. To infer variations in mitochondrial haplotypes, we sequenced a 644 bp region of the mitochondrial cytochrome c oxidase subunit I (COI) gene from 33 females collected in 2011 and 24 collected in 2016. PCR amplification was performed by using a pair of primers, LCO1490 and HCO2198 [27]. The PCR products were subjected to ExoSAP-IT clean-up and then sequenced by Eurofins Genomics K.K.

A haplotype network was generated from the 644 bp sequences of COI in TCS v. 1.21 software [28], which calculates the minimum number of mutation steps by which the sequences can be jointed with greater than 95% confidence.

3. Results

(a). Rapid flux in sex ratio between 2011 and 2016

Our previous study showed that the sex ratio of M. desjardinsi adults collected in Matsudo was strongly female biased in 2011 (57 females : 7 males, p = 7.638 × 10–11 by EBT) [23]. The offspring of these females were also strongly female biased (total of 1575 females : 516 males; p < 2.2 × 10–16 by EBT; electronic supplementary material, table S1) [23]. In 2016, the sex ratio was still female biased (80 females : 49 males; p = 0.008 by EBT), but the bias was less extreme than in 2011 (p = 8.311 × 10–5 by Fisher's exact probability test). The sex ratio in the offspring of these females was close to 1 : 1 (1189 females : 1087 males; electronic supplementary material, table S1), although with a slight bias towards females (p = 0.034 by EBT).

(b). The absence of male killing despite a consistently high prevalence of Spiroplasma in 2016

In 2011, of 34 wild-caught females, 25 (73.5%) were positive for Spiroplasma [23]. Among the 25 broods produced by the Spiroplasma-positive females, 21 consisted of only females, two were female biased (p < 0.05 by EBT) and the remaining two had sex ratios that did not deviate significantly from 1 : 1 (p > 0.05; figure 1a; electronic supplementary material, table S1).

In 2016, we found that 21 out of 33 wild-caught females (63.6%) were positive for Spiroplasma (no significant difference in the frequency of infection between 2011 and 2016; p = 0.4372 by Fisher's exact probability test). Nevertheless, all females produced both males and females, irrespective of Spiroplasma infection; among the 21 broods produced by Spiroplasma-infected females, 20 had sex ratios that did not deviate significantly from 1 : 1 (p > 0.05 by EBT); one brood (no. 15) had a female-biased sex ratio (p = 0.0429) (figure 1b; electronic supplementary material, table S1). Although the survival rates (i.e. number of adults emerging divided by number of eggs laid) of almost all broods produced by infected females in 2011 were less than 50% [23], those of all broods in 2016 were greater than 50%, regardless of the infection status of the mothers (electronic supplementary material, table S1). In 2011, the survival rates of broods produced by Spiroplasma-infected females were significantly lower than those by uninfected females (χ21 = 138.71, p < 2.2 × 10–16 by LR test). By contrast, Spiroplasma infection did not affect the survival rates of broods in 2016 (χ21 = 1.0617, p = 0.3028 by LR test).

(c). Appearance of male killing through introgression via 2011 males

In the crossing experiments, the origin of males (2011 or 2016) strongly affected the sex ratio of F1 broods (figure 2b). The proportions of male offspring were significantly lower when females were crossed with 2011 males than when they were crossed with 2016 males (χ21 = 10.217, p = 1.392 × 10−3 by LR test; figure 2b). All females crossed with 2016 males produced offspring with a nearly 1 : 1 sex ratio, with the exception of one (female no. 11, crossed with male no. 6), which produced a female-biased brood (16 females : 4 males; p < 0.05 by EBT) (electronic supplementary material, table S2). Among nine broods produced by females that were crossed with 2011 males, on the other hand, two consisted of only females. Among the remaining seven broods, consisting of both males and females, two were significantly female biased (electronic supplementary material, table S2). Furthermore, among the F2 and F3 offspring produced by females consecutively crossed with 2011 males, five broods consisted of only females (figure 2c; electronic supplementary material, table S2). All females were positive for Spiroplasma, with the exception of a female that was crossed with a 2011 male in the second generation (electronic supplementary material, table S2).

(d). The presence of the same Spiroplasma strain

Among 33 females collected in 2016, 21 were PCR positive for the Spiroplasma-specific spoT gene. Direct sequencing of the PCR products (513 bp) of 21 samples revealed that they were all identical to those of the 2011 samples (accession number: LC127423). Eighty offspring produced by 20 Spiroplasma-positive females (two daughters and two sons per brood) were PCR positive for spoT, suggesting that Spiroplasma was transmitted to both sons and daughters.

(e). Maintenance of mitochondrial haplotype variation

The 57 individuals analysed (n = 33 for 2011 samples; n = 24 for 2016 samples) were categorized into nine mitochondrial haplotypes (hap 1–9; accession numbers LC360405 to LC360413) on the basis of a partial sequence (644 bp) of the COI region. Among five haplotypes (hap 1, 2, 4, 5 and 6) shared by the 2011 and 2016 samples, hap 4 was present in the largest number of individuals (13 in 2011 and 12 in 2016) and was placed at the centre of the star-like topology generated by the haplotype network (figure 3). Haplotype frequencies did not differ significantly between 2011 and 2016 (electronic supplementary material, table S3; p = 0.8336 by Fisher's exact probability test).

Figure 3.

Figure 3.

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Haplotype spanning networks of Mallada desjardinsi collected in (a) 2011 (n = 33) and (b) 2016 (n = 24). Each circle represents a single haplotype with an area proportional to the sample size. Shaded areas, Spiroplasma-positive samples; open areas, Spiroplasma-negative samples. Cross-bars represent numbers of nucleotide substitutions.

4. Discussion

Here, we demonstrated a rapid change in sex ratio in an M. desjardinsi population: the proportion of males among captive lacewings was 10.9% in 2011 [23], but it had reached 38.0% by 2016. Total offspring produced by the captive females also demonstrated a similar increase in male ratio, from 24.7% in 2011 to 47.8% in 2016; these values more likely represent the real sex ratios of the population: the consistently smaller male ratio in captive adults than in their offspring probably reflects behavioural differences between the sexes (e.g. males being more active and thus difficult to capture than females).

Although high frequencies of infection with Spiroplasma were consistently observed in 2011 and 2016 (73.5 and 63.6%), all the females caught in 2016 produced male offspring. This suggests that in 2016, Spiroplasma was barely inducing male killing. Partial replacement of the genome of 2016 with that of 2011 by backcrossing restored the male-killing expression of Spiroplasma in some, but not all, of the lineages in the F1, F2 and F3 generations. The contrasting difference in sex ratio between the backcrossed and non-backcrossed offspring suggests that nuclear suppressors against male killing existed preferentially in the 2016 genome and that suppression is likely to have occurred zygotically, rather than maternally. However, the detailed genetic nature (i.e. mode of inheritance) of the suppressor or suppressors remains elusive. It is possible that there are multiple suppressors with different characteristics at different loci. The presence of suppressors in the 2011 line with low frequency cannot be completely ruled out, but it is highly unlikely that suppressor could emerge in the Spiroplasma-free laboratory line.

In conclusion, our results strongly suggest that nuclear suppressors against a cytoplasmic male killer had experienced a selective sweep and had become nearly fixed in the population within 5 years. In support of this notion, there was no significant difference in mitochondrial DNA diversity between individuals collected in 2011 and 2016; the finding of a significant difference would have supported the alternative notion that a particular matriline with a non-male-killing Spiroplasma had spread in the population.

On the basis of past temperature data for Chiba Prefecture obtained from the Japan Meteorological Agency and the effective cumulative temperature of M. desjardinsi [29], we infer that spread of the nuclear suppressors has been achieved within fewer than 30 generations (electronic supplementary material, table S4). It is likely that the M. desjardinsi population already had suppressors against male killing in 2011, albeit at a low frequency: 4 out of 25 Spiroplasma-infected captive females produced sons, although in varying numbers (electronic supplementary material, table S1). However, our previous finding that one of these four females produced Spiroplasma-free sons (table S5 in [23]) implies that there is another mechanism that suppresses male killing by causing selective transmission of Spiroplasma to daughters. On the other hand, the Spiroplasma-infected females collected in 2016 produced sons and daughters that were all Spiroplasma positive, suggesting that Spiroplasma was transmitted to both sexes. Therefore, it is likely that the suppression of male killing by selective transmission to daughters—if it exists—is very rare in M. desjardinsi. We speculate that zygotic suppression of male killing occurs by interfering directly with the as-yet unknown mechanisms of Spiroplasma-induced male killing.

The rapid spread of suppressors of Spiroplasma-induced male killing in the lacewings is the second documented case, the first being that found in the butterfly H. bolina, in which suppressors of Wolbachia-induced male killing spread on a Samoan Island within 5 years [16]. These two similar case studies of distinct endosymbionts and distinct host taxa demonstrate that this type of event, which was originally predicted by William D. Hamilton [30], is not unique to particular host–endosymbiont systems but may occur in diverse systems that have sex ratio distorters. The occurrence of endosymbionts with seemingly no phenotypic effects may be the consequence of the conflict between cytoplasmic elements and nuclear genes. We speculate that the spread of both cytoplasmic elements and the nuclear suppressors against them can be so rapid [16,17,31] that we may only rarely observe the dynamic turnover that may be occurring repeatedly in diverse species.

We demonstrated here that lacewing hosts rapidly evolved suppressors against selfish reproductive manipulation by a Spiroplasma endosymbiont. If it is no longer causing male killing, in the absence of any other reproductive advantage the Spiroplasma is likely to decrease in prevalence in the lacewing population because of its potential costs to the host and its imperfect vertical transmission [23,32,33]. On the other hand, if Spiroplasma acquires traits that offset the male-killing suppression or if it confers fitness advantages on its hosts, it may be maintained in the host population. Our anticipated follow-up observations of the population dynamics of M. desjardinsi and Spiroplasma may bring new discoveries in the years to come.

Supplementary Material

Infection status of females collected in 2011 and 2016, and sex ratios of their offspring
rspb20180369supp1.xlsx (16.6KB, xlsx)

Supplementary Material

Sex ratios of offspring produced by Spiroplasma-infected 2016 females crossed with 2011 males (outbred) or 2016 males (inbred)
rspb20180369supp2.xlsx (14.7KB, xlsx)

Supplementary Material

Mitochondrial haplotypes and Spiroplasma infection status of females collected in 2011 and 2016
rspb20180369supp3.xlsx (10.7KB, xlsx)

Supplementary Material

Estimation of voltinism in the green lacewing Mallada desjardinsi in Matsudo
rspb20180369supp4.xlsx (11.7KB, xlsx)

Acknowledgements

We thank three anonymous referees for valuable comments on our manuscript.

Ethics

Collection of M. desjardinsi, a non-endangered insect, made in Chiba University campus, does not violate any laws.

Data accessibility

Raw data of M. desjardinsi samples collected in Matsudo were deposited in the Dryad Digital Repository: (http://dx.doi.org/10.5061/dryad.pg6qq10) [34].

Authors' contributions

M.H. and D.K. conceived the study. M.H. and D.K. participated in the design of the study. M.H. bred the insects and performed the genetic experiments and statistical analyses. D.K. performed the molecular analyses and coordinated the study. M.H., M.N. and D.K. drafted the manuscript. All authors gave final approval for publication.

Competing interests

We have no competing interests.

Funding

This study was supported partially by JSPS KAKENHI grant (no. 17J04148) to M.H. and (no. 16K08106) to D.K.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

  1. Hayashi M, Nomura M, Kageyama D. 2018. Data from: Rapid comeback of males: evolution of male-killer suppression in a green lacewing population Dryad Digital Repository. ( 10.5061/dryad.pg6qq10) [DOI] [PMC free article] [PubMed]

Supplementary Materials

Infection status of females collected in 2011 and 2016, and sex ratios of their offspring
rspb20180369supp1.xlsx (16.6KB, xlsx)
Sex ratios of offspring produced by Spiroplasma-infected 2016 females crossed with 2011 males (outbred) or 2016 males (inbred)
rspb20180369supp2.xlsx (14.7KB, xlsx)
Mitochondrial haplotypes and Spiroplasma infection status of females collected in 2011 and 2016
rspb20180369supp3.xlsx (10.7KB, xlsx)
Estimation of voltinism in the green lacewing Mallada desjardinsi in Matsudo
rspb20180369supp4.xlsx (11.7KB, xlsx)

Data Availability Statement

Raw data of M. desjardinsi samples collected in Matsudo were deposited in the Dryad Digital Repository: (http://dx.doi.org/10.5061/dryad.pg6qq10) [34].

Articles from Proceedings of the Royal Society B: Biological Sciences are provided here courtesy of The Royal Society

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