copulatory mate guarding - Wiley Online Library

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Nov 8, 2017 - Keiko Oku, College of Life and Environmental. Sciences ... Cox, 2016; Han, Jablonski, & Brooks, 2015; Hazlett & Rittschof, 2000;. This is an ...
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Received: 27 July 2017    Revised: 29 October 2017    Accepted: 8 November 2017 DOI: 10.1111/eth.12710

R E S E A RC H PA P E R

Female response to predation risk alters conspecific male behaviour during pre-­copulatory mate guarding Keiko Oku1,2

 | Erik H. Poelman1 | Peter W. de Jong1 | Marcel Dicke1

1 Laboratory of Entomology, Wageningen University, Wageningen, The Netherlands

Abstract

2 National Agriculture and Food Research Organization, Agricultural Research Center, Tsukuba, Ibaraki, Japan

Mating behaviour often increases predation risk, but the vulnerability within mating

Correspondence Keiko Oku, College of Life and Environmental Sciences, University of Exeter, Penryn, UK. Email: [email protected]

mite Tetranychus urticae, males engage in pre-­copulatory mate guarding because only

Funding information Japan Society for the Promotion of Science, Grant/Award Number: 24-258; Canon Foundation in Europe

males, (ii) whether the vulnerability to the predator differs between sexes within the

Editor: T. Tregenza

pairs differs between the sexes. Such a sex difference is expected to lead to differences in responses to predation risk between the sexes. In the two-­spotted spider the first mating results in fertilisation. We investigated (i) whether pre-­copulatory pairs are more conspicuous to the predatory mite Phytoseiulus persimilis than solitary fepre-­copulatory pair, (iii) whether each sex of T. urticae responds to predation risk during pre-­copulatory mate guarding and (iv) whether T. urticae’s response to predation risk affects predator behaviour. Because T. urticae females are immobile during pre-­ copulatory mate guarding, we observed male behaviour to evaluate effects of predation risk. We found that the predators detect more pre-­copulatory pairs than solitary females and that more females than males of the pre-­copulatory pairs are preyed upon by the predators. The preference of spider mite males for pre-­copulatory pairs versus solitary females was affected by whether or not the female had been exposed to predators during development. Male T. urticae exposed to predation risk did not alter their behaviour. These results suggest that only the most vulnerable sex, that is the female, responds to predation risk, which modifies male behaviour. Regardless of T. urticae females’ experience, however, P. persimilis detected more T. urticae pre-­copulatory pairs than solitary females, suggesting that pre-­copulatory mate guarding itself is dangerous for T. urticae females when these predators are present. We discuss our results in the context of sex-­dependent differences in predation risk. KEYWORDS

pre-copulatory pair, predatory mite, sex difference, solitary female, spider mite

1 |  INTRODUCTION

change behaviour in response to predation risk (Acharya & McNeil, 1998; Koga, Backwell, Jennions, & Christy, 1998; Mathis & Hoback,

Prey organisms alter their behaviour upon exposure to predators or their

1997; Sih, Krupa, & Travers, 1990), this is not always the case. Examples

cues to avoid predation (Lima & Dill, 1990). Mating behaviour often in-

have been described where the behavioural responses to predation risk

creases predation risk of both males and females (Andersson, 1994;

are sex-­dependent (Bernal, Rand, & Ryan, 2007; Curlis, Macklem, Davis,

Magnhagen, 1991). Although many studies showed that both sexes

& Cox, 2016; Han, Jablonski, & Brooks, 2015; Hazlett & Rittschof, 2000;

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors Ethology Published by Blackwell Verlag GmbH Ethology. 2017;1–9.

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Magurran & Nowak, 1991; Wormington & Juliano, 2014). In many organisms, one sex is more vulnerable to predators than the other sex while mating (Kojima, Sugiura, Makihara, Ishikawa, & Takanashi, 2014; Raghuram, Deb, Nandi, & Balakrishnan, 2015; Su & Li, 2006). Such sex differences can impose different costs of mating behaviour on each sex. When costs of mating behaviour differ between sexes, the selective pressure on predation avoidance is different between the sexes. Consequently, responses to predation risk become sex-­dependent. Furthermore, it is possible that only one sex responds to predation risk, which affects the behaviour of the other sex (Dill, Hedrick, & Fraser, 1999; Evans, Kelly, Ramnarine, & Pilastro, 2002; Su & Li, 2006). In species where females are more often preyed upon by predators than males, for example, females exposed to predators become less receptive to male courtship, which results in reduced male courtship frequency (Su & Li, 2006). Thus, observed behavioural responses may not always involve direct responses to the predator by both sexes and may involve a more

F I G U R E   1   Pre-­copulatory mate guarding of Tetranychus urticae

complex interaction between mates. In mating behaviour, pre-­copulatory mate guarding is a male mating strategy to ensure paternity (Conlan, 1991; Jormalainen, 1998; Thornhill & Alcock, 1983), which has evolved in organisms in which female sexual receptivity is limited to a short time interval (Ridley, 1983) or in which the first male’s insemination has an advantage in sperm competition (Yasui, 1988). Pre-­copulatory pairs often attract more attention from predators than single individuals (Cothran, 2004; Ward, 1986), and the vulnerability within pre-­copulatory pairs is not equal for the two sexes (Cothran, 2004). Such a sex difference in vulnerability may select for different responses to predation risk between the two sexes. To our knowledge, however, no study has examined in any species how each sex responds to predation risk during pre-­copulatory mate guarding. In spider mites of the genus Tetranychus (Acari: Tetranychidae), adult males guard quiescent deutonymph females, the stage immediately before adult emergence and sexual maturation (Potter, Wrensch, & Johnston, 1976; Satoh, Yano, & Takafuji, 2001; Figure 1), because only the first mating results in fertilisation unless interrupted (Helle, 1967; Oku, 2008; Potter & Wrensch, 1978; Satoh et al., 2001). During pre-­ copulatory mate guarding, male spider mites are mobile, while females are immobile because of their quiescent state. Thus, if there is a predator, the vulnerability to predation is expected to differ between the sexes and to be higher in females. Predatory mites (Acari: Phytoseiidae), which are major natural enemies of spider mites (McMurtry, Huffaker, & Van de Vrie, 1970), have no eyes and use chemical cues during for-

the predatory mite Phytoseiulus persimilis than solitary females, (ii) whether the vulnerability to the predators differs between the sexes within the pre-­copulatory pair and (iii) whether each sex of T. urticae responds to predation risk during pre-­copulatory mate guarding. We observed behaviour of male spider mites based on the phenomenon that they first approach pre-­copulatory pairs rather than solitary females. If pre-­copulatory pairs are more conspicuous to the predators than solitary females, and furthermore, if females within the pre-­ copulatory pairs are more vulnerable to the predators than males, female spider mites are expected to respond to predation risk more clearly than males. In order to understand prey–predator interactions, moreover, it is essential to include both aspects of prey and predators in the interactions (Lima, 2002; Sih, 1984). Allowing predators to behave in response to prey behaviour might change our expectations of prey behaviour (Lima, 2002). Thus, we also investigated whether T. urticae response to predation risk affects predator behaviour. Here, we report and discuss a contraction that sex-­dependent behavioural responses to predation risk can occur in organisms which males employ pre-­copulatory mate guarding, whereas the response to predation risk does not affect predator behaviour.

aging (De Boer & Dicke, 2006; Sabelis & Van de Baan, 1983). Previous

2 | MATERIALS AND METHODS

studies revealed in Tetranychus urticae and Tetranychus kanzawai that

2.1 | Study species

when a pre-­copulatory pair and a solitary female are present in the same patch under no predation risk, conspecific males first approach

Individuals of the two-­spotted spider mite T. urticae (Acari:

the pre-­copulatory pair rather than the solitary female (Oku, 2009; Oku

Tetranychidae) were reared on Lima bean plants (Phaseolus lunatus L.

& Shimoda, 2013; Oku, Weldegergis, Poelman, de Jong, & Dicke, 2015),

cv. Jackson Wonderbush) in a greenhouse (25 ± 5°C, 50%–70% relative

which is due to odour differences between pre-­copulatory pairs and

humidity, L 16: D 8). Phytoseiulus persimilis (Acari: Phytoseiidae) is a spe-

solitary females (Oku et al., 2015). If predatory mites show preference

cialist predator of Tetranychus spider mites (McMurtry & Croft, 1997).

similar to male spider mites, pre-­copulatory pairs are expected to alter

Predatory mites were kindly provided by Entocare C.V. (Wageningen,

emission of chemical signals in order to reduce predation risk. This may

The Netherlands) and Koppert Biological Systems (Berkel en Rodenrijs,

result in modifying behaviour of conspecific males.

The Netherlands) and reared for several generations in Petri dishes

In this study, we investigated (i) whether pre-­copulatory pairs

(90 mm in diameter, 15 mm in depth) containing P. lunatus leaves in-

of the two-­spotted spider mite T. urticae are more conspicuous to

fested by T. urticae in a climate chamber (25 ± 1°C, 50%–70% relative

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OKU et al.

humidity, L 16: D 8). Each Petri dish was sealed with Parafilm (Parafilm

examine whether the vulnerability to predators differs between the

M, American National Can, Chicago, IL; not a food source for mites) to

sexes within pre-­copulatory pairs, 55 T. urticae deutonymph females

prevent any mites from escaping from the Petri dishes.

were individually transferred to 55 leaf squares (10 × 10 mm) placed

The life cycle of T. urticae consists of egg, larva, quiescent larva,

on water-­saturated cotton wool in small Petri dishes. They were kept

protonymph, quiescent protonymph, deutonymph, quiescent deu-

for 1 day in a climate cabinet to develop into quiescent deutonymphs.

tonymph and adult stages. As T. urticae is arrhenotokous, virgin fe-

Then, one T. urticae adult male (2 days old) was introduced onto each

males can produce only sons (Boudreaux, 1963). Males arising from

leaf square and allowed to guard the female. After 2 h, one P. persimilis

virgin females were used in this study. To obtain adult males from

female that had been deprived of food for 2 days was introduced to

virgin females, 15–20 quiescent deutonymph females were randomly

each leaf square and observed for 30 min. We recorded which sex of

selected from the stock cultures. They were transferred with a fine

T. urticae was first attacked and then preyed upon by P. persimilis. To

brush to leaf discs, which were cut from expanded primary leaves of

determine whether the first attack of predators differed significantly

P. lunatus, placed on water-­saturated cotton wool. After adult emer-

from a 1:1 ratio between the sexes of T. urticae, the data were ana-

gence, the females were allowed to oviposit for 5 days in a climate

lysed using a binomial test in the statistical package R version 2.10.0

cabinet (25 ± 1°C, 50%–70% relative humidity, L 16:D 8). When the

(R Development Core Team, 2010). Furthermore, a chi-­square test in

male offspring resulting from these eggs developed into the quies-

the statistical software JMP version 11.0.0 (SAS Institute, 2013) was

cent deutonymph stage, they were transferred to new leaf discs. Adult

applied to compare the distribution of first attack and predation over

males of known age were obtained by this procedure.

the sexes of the spider mites.

2.2 | Experiments

2.2.3 | Does prior exposure of females to predation risk affect male behaviour?

2.2.1 | Does pairing increase detection by predators?

When a pre-­copulatory pair and a solitary female of spider mites

To examine whether pre-­copulatory pairs and solitary females of spider

are present in the same patch under no predation risk, conspecific

mites are equally well detected by predatory mites, two T. urticae qui-

males first approach the pre-­copulatory pair rather than the solitary

escent protonymph females, randomly selected from the stock culture,

female (Oku, 2009; Oku & Shimoda, 2013; Oku et al., 2015). To ex-

were transferred to 10 × 10-­mm leaf squares placed on water-­saturated

amine whether prior exposure of females to predation risk affects

cotton wool in Petri dishes (30 mm in diameter, 10 mm in depth, here-

male mate-­searching behaviour, 50 adult T. urticae females randomly

after referred to as “small Petri dish”). They were kept for 2 days in a

selected from the stock culture were transferred to each of 10 leaf

climate cabinet (25 ± 1°C, 50%–70% relative humidity, L 16:D 8) to

circles (50 mm in diameter) placed on water-­saturated cotton wool

develop into quiescent deutonymphs. Because the attractiveness of

in plastic containers (105 mm in diameter, 40 mm in depth) covered

quiescent deutonymph females to males slightly depends on the time

with a lid having a mesh hole (40 mm in diameter) in the centre. They

until adult emergence (Oku & Saito, 2014), females of similar ages were

were allowed to oviposit for one day. After removing the females, the

paired on each leaf square. Then, one T. urticae adult male (2 days old,

leaf circles with eggs were kept in a climate cabinet for 5 days. All

i.e., 2 days since the final moult) was introduced onto each leaf square

eggs hatched during the 5 days. Subsequently, one P. persimilis adult

and allowed to guard either female. After 2 h, one P. persimilis female

female randomly selected from the stock culture was introduced to

that had been deprived of food for 2 days was added to each leaf square

half of the leaf circles (“with predator exposure”), while the remaining

and observed for 30 min. We recorded whether P. persimilis females first

leaf circles served as controls (“without predator exposure”). For each

approached the pre-­copulatory pair or the solitary female. When P. per-

treatment, after 2 days, two deutonymph females were transferred

similis females escaped from leaf squares, they were gently returned to

to new 10 × 10-­mm leaf squares on cotton wool in small Petri dishes.

the original leaf square. Each leaf square with one pre-­copulatory pair

They were now present in the absence of predators. On the next day,

and one solitary female was used only once. The cases where P. persimi-

one adult male (2 days old) was glued on the back of one female to

lis females approached neither individual within the 30-­min observation

keep him at the natural guarding position (Figure 1), as female expe-

period were excluded from the data set. This experiment was replicated

rience of predation risk may not let the male stay there. The other

three times with 19–33 trials per replicate, each using a naïve predatory

female remained solitary. Only glue (a water-­soluble glue, “Bondo”;

mite female in the choice test. To determine whether pre-­copulatory

Konishi Company, Osaka, Japan) was applied onto the back of the

mate guarding affected prey detection by predators, the data were ana-

solitary female on each leaf square. Because the glue is too sticky to

lysed using a replicated G test (Sokal & Rohlf, 1995).

apply on spider mites, it was diluted with water before use. This glue has no effects on behaviour of spider mites (Oku, 2009). One hour after gluing the male onto the quiescent female, another T. urticae

2.2.2 | Do the sexes differ in vulnerability to predators when paired?

male was introduced onto each leaf square and observed for 30 min.

During pre-­copulatory mate guarding, male spider mites are mobile,

cases where males approached neither individual within the 30-­min

while females are immobile because of their quiescent state. To

observation period were excluded from the data set. This experiment

We recorded which female was approached first by the male. The

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was replicated four times with 27–55 trials per replicate. Before de-

Then, one P. persimilis female was introduced to half of the leaf circles

termining the effects of prior exposure of female spider mites to pre-

(“with predator exposure”), while the remaining leaf circles served as

dation risk on behaviour of T. urticae males, the heterogeneity among

controls (“without predator exposure”). At the same time, T. urticae

replicates was investigated for each treatment group (Sokal & Rohlf,

deutonymph females, randomly selected from the stock culture, were

1995). When there was no significant heterogeneity, the replicated

transferred to 10 × 10-­mm leaf squares (one female per leaf square)

data were pooled and analysed using a chi-­square test to access the

placed on water-­saturated cotton wool in small Petri dishes. On the

effect of treatment.

next day, males were individually transferred to leaf squares with one quiescent deutonymph female (without predator exposure: n = 67;

2.2.4 | Does current predation risk affect male behaviour?

with predator exposure: n = 60) and observed after one hour. We recorded whether males guarded the female or not. The cases where males exited leaf squares and drowned during the one-­hour period

When females had been previously exposed to predation risk, con-

since their introduction were excluded from the data set. To deter-

specific males did not show preference for pre-­copulatory pairs and

mine the effect of male prior experience with predation risk on their

solitary females (see Section 3). To rule out a possible interpretation

own guarding behaviour, the data were analysed using a chi-­square

that males of pre-­copulatory pairs perceive cues related to predation

test.

risk from their partner and subsequently affect mate-­searching behaviour of conspecific males, two T. urticae quiescent deutonymph females were prepared on 10 × 10-­mm leaf squares placed on water-­ saturated cotton wool in small Petri dishes. At the same time, 10 adult

2.2.6 | Does prior exposure of females to predation risk affect predator behaviour?

T. urticae females, randomly selected from the stock culture, were

When T. urticae females had not been previously exposed to preda-

crushed in 100 μl of distilled water in microtubes. After centrifuging

tion risk, predatory mites found more T. urticae pre-­copulatory pairs

the mixture, the supernatant was collected as “spider mite extract”.

than solitary females (see Section 3). To examine whether prior ex-

Because spider mites recognise cues from injured conspecifics as

posure of female spider mites to predation risk affects behaviour

predation risk (Grostal & Dicke, 1999; M. Dicke, unpublished data;

of predatory mites, T. urticae females were exposed to P. persimi-

Supporting Information), this spider mite extract was used as indicator

lis during their developmental period in the same manner as de-

of predation risk. We applied 10 μl of the spider mite extract on each

scribed above. For each treatment (i.e., with and without predator

leaf square in the presence of two T. urticae quiescent deutonymph

exposure), two quiescent deutonymph females were prepared on

females. Then, one T. urticae adult male (2 days old) was glued on the

10 × 10-­mm leaf squares placed on water-­saturated cotton wool in

back of one of the two females. Only glue was applied on the back

small Petri dishes. Then, one T. urticae adult male (2 days old) was

of the other female. One hour later, another T. urticae male was in-

glued on the back of one of these females. Only glue was applied

troduced to each leaf square and observed for 30 min. We recorded

on the back of the other female on each leaf square. One hour after

which female the male approached first. Cases where males did not

gluing, one P. persimilis female that had been deprived of food for

approach an individual within the 30-­min observation period were ex-

2 days was introduced to each leaf square and observed for 30 min.

cluded from the data set. This experiment was replicated three times

We recorded which spider mite was approached by P. persimilis first.

with 21–37 individually tested males per replicate. A new arena with

The cases where P. persimilis females approached neither individual

new females was used for each male tested. To determine whether

with the 30-­min observation were excluded from the data set. This

current predation risk affected mate-­searching behaviour of T. urticae

experiment was replicated four times with 26–60 trials per repli-

males, the data were analysed using a replicated G test (Sokal & Rohlf,

cate. Before determining the effects of prior exposure of female

1995).

spider mites to predation risk on behaviour of predators, the heterogeneity among replicates was investigated for each treatment

2.2.5 | Does prior exposure of males to predation risk affect their guarding behaviour?

group (Sokal & Rohlf, 1995). When heterogeneity was detected, the data were analysed using chi-­square tests for each replicate separately. Moreover, to examine whether individuals show a prefer-

To examine whether prior exposure of males to predation risk affects

ence for either T. urticae females with a male glued on their back

their willingness to guard females, 10 T. urticae adult females from the

or solitary females in their behaviour, G tests were applied for each

stock culture were transferred to each of four leaf circles placed on

choice test.

cotton wool in plastic containers with a lid having a mesh hole. They were allowed to oviposit for one day. Because predatory mites, including P. persimilis, that specialise on spider mites prefer eggs over other stages (Blackwood, Schausberger, & Croft, 2001), the T. urticae eggs laid on the leaf circles served as food for P. persimilis (see below).

3 | RESULTS 3.1 | Does pairing increase detection by predators?

After removing the females from the leaf circles, 40 T. urticae adult

There was no heterogeneity in the results of the three replicates

males (1–2 days old) were transferred onto each of four leaf circles.

(Table 1). The pooled data show that pre-­copulatory pairs had a ca

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T A B L E   1   Results and statistical analysis of predator (Phytoseiulus persimilis) choice tests when offered pre-­copulatory pairs (“PCP”) versus solitary females (“SF♀”) of Tetranychus urticae P. persimilis choice

Individual G test

Replicate no.

PCP

SF♀

Neither

1

19

5

9

Replicated G test

G-­value

df

p-­Value

8.708

1

.003

Heterogeneity Pooled

2

14

5

0

4.439

1

.035

3

22

4

1

13.719

1