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University of Groningen

The role of male courtship behaviour in prezygotic isolation in NasoniaPeire Morais, Aitana

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Publication date:2007

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Citation for published version (APA):Peire Morais, A. (2007). The role of male courtship behaviour in prezygotic isolation in Nasonia: Do waspsfinish what bacteria started?. s.n.

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4. Genetics of courtship behaviour:

A selection experiment inNasonia vitripennis

(Hymenoptera: Pteromalidae)

Aitana Peire, Albert Kamping, Louis van de Zande and

Leo W. Beukeboom

Peire-Morais, A., A. Kamping, L. van de Zande and L.W. Beukeboom. 2003. Genetics of courtship behaviour: a selection experiment in

Nasonia vitripennis (Hymenoptera: Pteromalidae). Proc. Exper. Appl. Entomol. NEV Amsterdam 14: 81-85.

04-Genetic of courtship 19/2/07 09:41 Página 71

Abstract

The three sibling species from the Nasonia group (N. vitripennis, N.longicornis, N. giraulti) broadly show similar courtship behaviourpatterns but with species-specific variations. Previous studies onhybrids between N. vitripennis and N. longicornis have shown atendency of hybrid males to behave as the (grand) paternal species. Itis not known whether this “grandfather effect” occurs within species.A selection experiment in N. vitripennis was performed to obtain lineswith extreme values for a particular behavioural character whichcould subsequently be used for studying its inheritance within thisspecies. Although in previous studies evidence for a genetic basis ofcourtship behaviour was found, the component under selection(headnod number) showed only a weak response.

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Introduction

In recently diverged species, the study of behaviour may explain howreproductive isolation evolved, or how it has been maintained. In theNasonia sibling species group, composed of N. vitripennis, N. giraultiand N. longicornis, infection with different strains of Wolbachiabacteria cause the reproductive isolation, through cytoplasmicincompatibility in interspecific crosses (Bordenstein and Werren,1998). However, after treatment with antibiotics, and despite somehybrid breakdown, viable and fertile offspring can be obtainedfrom interspecies crosses (Breeuwer and Werren, 1995).

Courtship and mating behaviour of Nasonia has been wellstudied (Van den Assem, 1986; Van den Assem and Werren, 1994). Amale mounts on top of a female and starts the courtship, performingstrong movements with the head that coincide with the release ofpheromones. These movements are the so-called head nods, and areperformed in consecutive series. Other components come along withthe courtship display such as “feet rubbing” (legs movement“sweeping” the females eyes), and “minus nods” (inconspicuous headmovements). The three species show many similarities in courtshipcomponents, but some differences are species-specific. For example,the mean number of head nods in the first nods series is species-specific, and feet rubbing and minus nods are only present in N.longicornis (Van den Assem and Werren, 1994).

The study of N. vitripennis x N. longicornis hybrids’ behaviourshowed a peculiar outcome. As expected by general mendelian laws ofinheritance, hybrid behaviour was intermediate to both parentalspecies, but it showed a significant bias towards the grandfathers’species in both reciprocal crosses (“grandfather effect”, Beukeboomand Van den Assem, 2001). This cannot be explained by cytoplasmicfactors, because in that case it would show a bias towards thegrandmothers’ species behaviour. It has not yet been establishedwhether this grandfather effect is restricted to hybrids, or if it also occurs

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The role of male courtship behaviour in prezygotic isolation in Nasonia...


in pure species. This would be of special interest, since “… thepossibility that the maternal and paternal genomes of hymenopteranfemales are nonequivalents could prompt a rethink of three decades oftheoretical models on the evolution of social behavior in ants, waspsand bees” (Haig, 1988). To answer this question, behavioural lines withquantitatively different characters are needed. In Nasonia vitripennis,most natural lines show similar behavioural features (chapter 2). It istherefore necessary to artificially generate divergent behavioural lines,to subsequently cross them and test for the grandfather effect. For thispurpose, a selection experiment was performed in N. vitripennis. Thecharacter under selection was the number of head nods in the firstseries. Four lines were selected for a high number of nods, and four fora low number of nods for four generations.

Material and Methods

Collection and culturingThe source population was established from a mixture of five fieldlines collected from three different nest boxes and one bait at theHoge Veluwe Park, and one bait from the Spanderswoud in Bussum,all in the central part of The Netherlands. It was started by puttingtogether 10 male and 30 female pupae from each of the field lines inthe 4th generation after collection. The emerged wasps matedamongst each other and were subsequently maintained as a massculture by transferring 100 mated females every generation. Nasoniawasps are easily cultured on Protocalliphora pupae. Generation timeis 14 days at 25º C. Like all Hymenoptera, Nasonia has haplodiploidsex determination, i.e., males are haploid and arise from unfertilisedeggs, and females are diploid and develop from fertilised eggs.Therefore, the sibship of a virgin female is all male, and the sibship ofa mated female consists of females and males.

4. Genetics of courtship behaviour: A selection experiment in Nasonia...

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Experimental design47 females were randomly collected from the mass culture as pupae,and individually hosted as virgin. They were kept alive in therefrigerator till their offspring emerged. Male offspring were collectedfrom the host pupae in the black pupal stage, kept in groups of fiveuntil emergence, and then isolated. The courtship behaviour of onerandomly chosen son per female was scored one day after emergenceunder standardised conditions, as described in Beukeboom and Vanden Assem (2001). The 8 most extreme males were selected (4 highestand 4 lowest number of head nods). They were mated with theirmothers to yield an inbred sibship, from which females were in turnhosted as virgins. Again, the 8 most extreme male offspring wereselected to produce the next generation for the high and low lines,respectively. When the mother was not available anymore, theselected male was mated back to an aunt. We performed a total offour such selection rounds. After 4 generations of selection 25 maleswere scored per selection line.

Molecular analysisFor establishing the inbred level, Amplified Fragment LengthPolymorphism (AFLP) analyses were performed using Mse-CAC withEco-ACA, and Mse-CAT with Eco-ACA as selective primers. Sixindividuals of the source population and eight individuals each of onelow and one high line were analysed using Mse-CAC with Eco-ACAprimers. Seven individuals of the source population and tenindividuals each of the same low and high line were analysed usingMse-CAT with Eco-ACA primers. The band sharing coefficientbetween individuals was calculated as Sxy = 2nxy / (nx + ny), where nxand ny are the number of fragments present in individuals x and yrespectively, and 2nxy is the number of fragments shared by x and y.Mean band sharings similarity was obtained by averaging pairwisecomparisons within and between treatment groups (see Kappe et al.,1997).

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StatisticsThe realised heritability, defined as the ratio between the mean of theselected parents (in our case grandfathers) divided by the mean of theresulting sibship, is a measure for the efficiency of selection. It isvirtually identical to the narrow sense heritability (Hartl and Clark,1997). It was calculated for every round of selection. Mann-WhitneyU tests were performed to test for differences in behaviour betweenthe high and the low lines in each generation of selection.

Results

The mean number of head nods before selection was 5.72 with arange of 2-9, and a standard error of 0.06 (n=47, fig. 1). As shown infigure 2, high and low lines diverged in head nods numbers after 4generations of selection. This divergence is maximal in the secondgeneration of selection (average of low lines 5.03, SE 0.02 and averageof high lines 6.25, SE 0.02), drops in the third generation, andbecomes again significant in the fourth generation of selection (table1). After 4 generations of selection the lowest and highest values ofhead nods have disappeared in the low and the high lines (figure 1).

The realised heritability values show a weak response toselection, with no mean values significantly different from zero (table2). Overall heritabilities, from generation 0 to 4, were 0.09 for the highand 0.13 for the low lines (table 2).

The AFLP analyses yielded 28 scorable bands ranging from 104to 478 bp for Mse-CAC with Eco-ACA, and 28 scorable bands rangingfrom 106 to 447 base pairs (bp) for Mse-CAT with Eco-ACA. Theaverage band sharing coefficients were calculated. Both within line andbetween lines, the level of variation is low (average band sharing si-milarity around 0.9), although there is a tendency of decreasingsimilarity between lines, especially between treatment groups (table 3).


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One high line was lost after one generation of selection, and one lowline was lost after three generations of selection. In addition, as theselection experiment advanced, the mortality rate and the diapausefrequency increased.

Figure 1. Distribution of the number of headnods in the first series in thesource population before selection, and in the last generation of

selection (4th generation) for the high and the low lines.

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Figure 2. Mean numbers of headnods in the first series for the different linesof selection across generations. Open symbols correspond tofamilies selected for a low number of headnods, and closedsymbols to families selected for a high number of headnods. Thedashed line indicates the source population in generation 0.


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Table 1. Average number of headnods in the first series. The low and the high lines for the different generations of selection;

sample sizes and P values for the Mann-Whitney U-tests.

Table 2. Realised heritabilities. Calculated as HR=R/S (Hartl and Clark, 1997). SE is the standard

error of the mean.

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Table 3.Mean band sharing similarity (s

_) obtained by AFLP analysis.

Numbers in brackets represent standard errors.

Discussion

After four rounds of selection for headnod numbers in courtship ofNasonia vitripennis, behavioural differences between lines wereobtained. However, the response to selection was weak. The largestaverage difference was 1.22 head nods after two generations ofselection, decreasing to 0.42 head nods in the 4th generation ofselection. These results are consistent with previous studies that showlow heritability values for behavioural traits (Meffert and Regan, 2002).

Previous studies have shown a high repeatability for this trait(chapter 3). In addition, Beukeboom and Van den Assem (2001)showed that hybrid males had intermediate behaviour between bothparental species. Both results suggest that the trait is at least partiallygenetically determined. How can the absence of a strong response to


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selection be explained? First, there could be a lack of genetic variationin the source population. This population was established frommixing five recently field-collected populations, in which each one ofthem originated from parasitised fly pupae in a nest box or bait.Unfortunately, the number of founding females is not known, butthere must have been at least one inseminated female per nest or bait,because both male and female offspring emerged from the fly pupae.The preliminary molecular analysis indicates a low level of geneticvariation in the source population. Although the selection regime, thatcomprises mother-son crosses every other generation, increaseshomozygosity by 50% each generation, it may have remainedundetected by the molecular analysis due to the already low level ofgenetic variation. However, the tendency of increasing differentiationbetween treatment groups as compared to within treatment groupsmay be a reflection of an inbreeding effect. In addition, a highermortality and diapause frequency was observed for the selection linesin the course of the experiment, which could also be the result of thehigh degree of inbreeding. If male survival and/or diapauseavoidance represents an opposing selective force to headnod number(at least in the present, genetically deplenished source population) itcould explain the disappearance of the initial response. Futureexperiments are aimed at finding or generating different behaviourallines in N. vitripennis to unravel the genetic architecture of courtshipbehaviour, and its role in reproductive isolation.

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