Male moths that shed their genital spines inside the female: A survey of male mating costs and material investment during copulation
Jaime Camacho-García 1 , Samuel Pineda 2 , Carlos Cordero Corresp. 3
1 Posgrado en Ciencias Biológicas, Instituto de Ecología, Universidad Nacional Autónoma de México, México, D. F., Mexico 2 Instituto de Investigaciones Agropecuarias y Forestales, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, Mexico 3 Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, México, D. F., México
Corresponding Author: Carlos Cordero Email address: [email protected]
In some insects, males produce costly ejaculates that reduce the typical sexual imbalance in potential reproductive rates thus influencing the mode and intensity of sexual selection. In Lepidoptera, in particular, males tend to invest heavily in mate searching, courtship and ejaculate production; furthermore, in some species males shed genital spines (deciduous cornuti) within the female genital tract during copulation. We studied experimentally the potential mating costs incurred by males of a moth with deciduous cornuti, Amorbia cuneana (Tortricidae). In the first experiment, we tested the prediction that mating costs result in a reduction in male longevity and survival rate. Our results did not support this prediction because virgin and mated males had similar longevity and survival rate. This result is somewhat surprising considering the substantial resource investment in mating found in the second experiment. In their first mating males invested in the ejaculate 9% of their body weight and transferred virtually all their deciduous cornuti; furthermore, few males were able to mate a second time (although we do not know if this was due to female rejection), these males produced smaller ejaculates and there was no evidence of cornuti regrowth. We hypothesize that reduced mating opportunities have selected for males that are ready to transfer large ejaculates at any moment and thus against the diversion of resources from reproductive tissues to somatic maintenance in unmated males; the transfer of the full set of deciduous cornuti in first copulations is consistent with this hypothesis.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 1 Male moths that shed their genital spines inside the female: A survey of male
2 mating costs and material investment during copulation
3
4 Jaime Camacho-García1
5 Samuel Pineda2
6 Carlos Cordero3
7
8 1 Posgrado en Ciencias Biológicas, Instituto de Ecología, Universidad Nacional Autónoma de
9 México, Distrito Federal, México.
10 2 Instituto de Investigaciones Agropecuarias y Forestales, Universidad Michoacana de San
11 Nicolás de Hidalgo, Morelia, Michoacán, México.
12 3 Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma
13 de México, Distrito Federal, México.
14
15 Corresponding author: Carlos Cordero, [email protected]
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 16 ABSTRACT
17 In some insects, males produce costly ejaculates that reduce the typical sexual imbalance in
18 potential reproductive rates thus influencing the mode and intensity of sexual selection. In
19 Lepidoptera, in particular, males tend to invest heavily in mate searching, courtship and ejaculate
20 production; furthermore, in some species males shed genital spines (deciduous cornuti) within
21 the female genital tract during copulation. We studied experimentally the potential mating costs
22 incurred by males of a moth with deciduous cornuti, Amorbia cuneana (Tortricidae). In the first
23 experiment, we tested the prediction that mating costs result in a reduction in male longevity and
24 survival rate. Our results did not support this prediction because virgin and mated males had
25 similar longevity and survival rate. This result is somewhat surprising considering the substantial
26 resource investment in mating found in the second experiment. In their first mating males
27 invested in the ejaculate 9% of their body weight and transferred virtually all their deciduous
28 cornuti; furthermore, few males were able to mate a second time (although we do not know if
29 this was due to female rejection), these males produced smaller ejaculates and there was no
30 evidence of cornuti regrowth. We hypothesize that reduced mating opportunities have selected
31 for males that are ready to transfer large ejaculates at any moment and thus against the diversion
32 of resources from reproductive tissues to somatic maintenance in unmated males; the transfer of
33 the full set of deciduous cornuti in first copulations is consistent with this hypothesis.
34 INTRODUCTION
35 In several insects, males produce costly ejaculates that reduce the typical imbalance in potential
36 reproductive rates of males and females thus influencing the mode and intensity of sexual
37 selection (Bonduriansky, 2001; Scharf, Peter & Martin, 2013). Male Lepidoptera, in particular,
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 38 tend to invest heavily in each mating. There is evidence that courtship is costly (Wedell, 2010)
39 and copula duration is relatively long (Scott, 1972). Furthermore, ejaculates are relatively large,
40 chemically complex, and they are built from resources that are obtained only during the larval
41 stage (i.e. non-renewable resources) (Drummond, 1984; Watanabe, 2016). Additionally, in
42 several species males have genital spines in the endophallus that are shed (deciduous cornuti)
43 inside the female reproductive tract during copulation (Fig. 1; Cordero, 2010; Cordero & Miller,
44 2012; Anzaldo, Dombroskie & Brown, 2014; Cordero & Baixeras, 2015).
45 Previous experimental studies of male mating costs in Lepidoptera show that in some
46 species mating behaviour and ejaculate production negatively affect male longevity and
47 survivorship rate (Shapiro, 1982; Drummond, 1984; Cordero, 2000; Ferkau & Fischer, 2006;
48 Wedell, 2010; Caballero-Mendieta & Cordero, 2013); however, in other species no negative
49 effects have been found (Svärd, 1985; Oberhauser, 1989; Ferkau & Fischer, 2006; Janowitz &
50 Fischer, 2010; Takeuchi, 2012; Callado-Galindo et al., 2013). On the other hand, even in cases
51 where no effect on male survivorship exists, the typical decrease in ejaculate size observed in
52 consecutive copulations of multiple mated males could be costly since ejaculate size tends to be
53 positively correlated with male fitness due to its positive effect on the length of the post-copula
54 period of diminished female sexual receptivity (Sugawara, 1979; Wedell, 2005; Watanabe,
55 2016) and on female fecundity (Torres-Vila & Jennions, 2005; Duplouy & Hanski, 2015). To the
56 best of our knowledge, male mating costs have not been studied in species with deciduous
57 cornuti.
58 We studied mating costs in males of a moth with deciduous cornuti, Amorbia cuneana
59 (Walsingham, 1879) (Lepidoptera: Tortricidae: Sparganothinii). We compared the longevity and
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 60 survival rate of virgin and mated males, and estimated material investment in copulation by
61 measuring the weight of the ejaculate and the number of deciduous cornuti transferred by males.
62 MATERIALS AND METHODS
63 Raising Amorbia cuneana
64 Amorbia cuneana, commonly known as the “Western Avocado Leafroller", is distributed from
65 Mexico to Canada (Phillips-Rodriguez & Powell, 2007), its caterpillars feed on trees from
66 several families (Phillips-Rodriguez & Powell, 2007; Juárez-Gutiérrez et al., 2015), and it is
67 considered a pest of avocado (Urías-López & Salazar-García, 2008). The moths used in the
68 experiments originated in a culture from the Instituto de Investigaciones Agropecuarias y
69 Forestales of the Universidad Michoacana de San Nicolás de Hidalgo (Juárez-Gutiérrez et al.,
70 2015). Moths were reared in an insectary at the Instituto de Ecología, Universidad Nacional
71 Autónoma de México, in a 12h light:12h dark photoperiod. Egg masses and larvae were reared in
72 four-ounces polypropylene plastic containers (one egg mass per container). Larvae were fed an
73 artificial diet (soy flour 42.7 g, wheat germ 19 g, Wesson salt mixture 6.4, Sucrose 7.8 g, Sorbic
74 acid 0.6 g, Methil paraben 0.96, Agar 13.6 g, Ascorbic acid 3.6, Acetic acid 25% 720 μl,
75 Formaldehyde 10% 264 μl, Cholin chloride 7.3 g, Vitafor-A™ 52 g, plus 600 ml of distilled
76 water; modified from Rosas-García and Villegas-Mendoza, 2008) presented as small blocks of
77 approximately 0.7 0.7 0.7 cm, until pupation. Individual pupae were placed in one-ounce
78 polypropylene plastic containers individually marked. Adult emergence date was recorded,
79 moths were sexed and the length of their front left wing, a surrogate of body size frequently used
80 in Lepidoptera, was measured with a calliper. Adults were fed a 10% honey solution presented in
81 a piece of absorbent fibre.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 82 Survival costs experiment
83 Measuring the effect of mating on male longevity and survival rate
84 In this experiment the hypothesis tested was that mating, ejaculate transfer and cornuti shedding
85 inside the female are costly for males and that these costs produce an increase in mortality rate
86 and, in consequence, a reduction in longevity. The experiment was originally designed to
87 compare the effect of mating on male longevity and survivorship rate between three groups of
88 males. One group was kept virgin for life, the second group was mated once, and the third group
89 was meant to mate twice. However, the "twice-mated” treatment was dropped from the
90 experiment because only six out of 13 males mated a second time (however, some data of this
91 small sample are presented as preliminary observations); the males of the "twice-mated”
92 treatment that mated only once were excluded from the analyses. An additional group consisting
93 of virgin males that failed to mate in two consecutive days despite being exposed to a different
94 virgin female each night (hereafter, “unsuccessful” males) was included as a third treatment.
95 Experimental males were kept individually in 1-ounce containers. Copulations were
96 obtained by introducing virgin couples in 4-ounces containers in the dark at midnight. Couples
97 were periodically observed under a red light until mating or until 6 AM; if no mating occurred at
98 this time, the male was returned to his 1-ounce container and exposed to a different virgin female
99 the next midnight. Males of the “virgin” treatment were also transferred during one night (from
100 midnight to 6 AM) to 4-ounces containers. Since adult did not emerged in the same date,
101 treatments were gradually completed in a period of several days. After mating or after a second
102 unsuccessful mating attempt, “once-mated” and “unsuccessful” males, along with “virgin”
103 males, were kept in their individual 1-ounce containers with fresh food and water until death.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 104 The front left wing length and longevity of virgin, once-mated and unsuccessful males
105 were compared with one-way ANOVA conducted by using the SPSS software (IBM SPSS
106 Statistics for Windows, version 21.0). Survivorship curves of the three experimental groups were
107 analyzed with the Kaplan-Meier method and the log rank test (95% confidence interval) by using
108 the R software (R Core Team, version 3.2.0).
109 Measuring the amount of materials invested in ejaculates
110 Females that mated with males of the “survival costs” experiment were euthanized by freezing
111 immediately after copulation and kept frozen until dissection. After the female was thawed at
112 ambient temperature, the abdomen was separated from the rest of the body and the bursa
113 copulatrix (ductus bursae + corpus bursae + accessory bursae), containing the ejaculate and
114 deciduous cornuti (these are mostly restricted to the corpus bursae), was dissected out under a
115 stereoscope (Olimpus BX-51), and weighed in a microbalance (model Orion Cahn C-33);
116 subsequently the corpus bursae was opened and the number of deciduous cornuti counted (Fig.
117 1A). The bursa copulatrix of five virgin females was dissected out, weighed, and its average
118 weight subtracted from the weight of the bursa copulatrix of each mated female to obtain the
119 weight of the ejaculate, which, therefore, also includes the weight of the cornuti. After death, the
120 phallus of the mated males was dissected out to count the number of remaining cornuti.
121 Relative investment experiment
122 Interspecific comparisons in the investment made by males in ejaculates employ the ratio of
123 ejaculate weight divided by pre-mating male weight (i.e., relative material investment). Since in
124 the “survival costs” experiment male weight was not measured, another experiment was
125 performed in which virgin males (n = 8) were weighed one day after their emergence from the
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 126 pupa (in a Radwag AX 220/X electronic balance), and the weight of their first ejaculates and
127 number of cornuti deciduous shed was obtained following the procedures described for the
128 previous experiment; immediately after copulation, these males were euthanized by freezing, and
129 their phallus dissected out to count the number of remaining cornuti.
130 RESULTS
131 General observations
132 In the survival costs experiment there were not significant differences in the left wing length
133 (ANOVA: F2,50 = 1.43, P = 0.25) of virgin, once-mated and unsuccessful males (Table 1A); the
134 wing length of the six males that mated twice was also similar (Table 1A). Twenty-four out of 26
135 once-mated males copulated the first time they had opportunity, the other two males mated the
136 second time they were exposed to a female; 25 out of 26 males mated when they were eight days
137 old or less. The 12 males that failed mating were exposed to two different females in two
138 consecutive days.
139 Do virgin males live longer than mated males?
140 The mean longevity of virgin, once-mated and unsuccessful males (Table 1A) was not
141 significantly different (ANOVA: F2, 51 = 1.86, P = 0.17). The mean longevity of five males that
142 mated twice was also similar (Table 1A). In agreement with this result, there were not statistical
143 differences between the survivorship curves of the three groups of males (log rank Mantel-Cox
144 test: chi- square = 3.0, df = 2, P = 0.22; Fig. 2).
145 Amount of materials transferred to females during copulation
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 146 The average weight of the bursa copulatrix of virgin females was 0.75 mg (± 0.32, median =
147 0.74, min–max: 0.4–1.184, n = 5). This average was subtracted from the weight of the bursa
148 copulatrix of each experimental female to obtain a “corrected” ejaculate weight.
149 The corrected average ejaculate weight produced by the males of the “relative
150 investment” experiment represented an average investment of 9.1% (± 3.1%, 4.2%–14.6%, n =
151 8) of male body weight (Table 1B). The corrected average weight of the ejaculates transferred by
152 males of the “survival costs” experiment, whose body weight unfortunately was not measured,
153 was significantly larger (t = 2.48, p = 0.018, df = 32; Table 1A), suggesting that we could be
154 underestimating the relative investment. The weight of the second ejaculate produced by four
155 males that mated twice in the “survival costs” experiment (median = 0.9 mg, min–max: 0.66–
156 1.12; two second ejaculates were not measured) was between 40% and 50% of that produced
157 during first copulations (Table 1A).
158 The average number of deciduous cornuti transferred to females by males of the “relative
159 investment” experiment was similar to that measured in once-mated males of the “survival costs”
160 experiment (Table 1). Three lines of evidence indicate that most males shed all or most of their
161 deciduous cornuti during their first copulation. First, in the “survival costs” experiment, 19 out of
162 the 25 once-mated males dissected (76%) had no cornuti and the other six males (24%) had very
163 few cornuti attached to the endophallus (three males had 1, one had 3, one had 4, and one had
164 10). In the “relative investment” experiment, 7 out of 8 males had no cornuti on the endophallus
165 when dissected after mating, and the other male had only one cornuti left (this male transferred
166 26 cornuti during mating). Second, four twice-mated males shed all cornuti in their first
167 copulation (Table 1A) since they did not shed any during their second mating (with exception of
168 a fragment of cornuti found in one female) and had no cornuti on the endophallus upon
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 169 dissection. Finally, the mean numbers of cornuti shed during first copulations in both
170 experiments are similar to the 28 and 29 cornuti observed in two virgin males.
171 The correlation between ejaculate weight and number of deciduous cornuti was not
172 significant, neither in the “survival costs” experiment (r = 0.16, P = 0.45, n = 25) nor in the
173 “relative investment” experiment (Spearman r = 0.24, P > 0.5, n = 8).
174 DISCUSSION
175 Male Amorbia cuneana transfer large ejaculates to females in first copulations, equivalent to
176 9.1% of mean male body weight. Despite transferring a large amount of substances, mated males
177 had similar mean longevity and mortality rates than males kept virgin for life and males that for
178 unknown reasons did not mate despite being exposed to virgin females (Fig. 2). Although
179 previous studies show that in some Lepidoptera species mating behaviour and ejaculate
180 production have negative effects on male longevity and mortality rate (Shapiro, 1982; Cordero,
181 2000; Ferkau & Fischer, 2006; Wedell, 2010; Caballero & Cordero, 2013), our results are not
182 completely novel since in other lepidopterans no negative effects have been found (Svärd, 1985;
183 Oberhauser, 1989; Ferkau & Fischer, 2006; Janowitz & Fischer, 2010; Takeuchi, 2012;
184 Callado-Galindo et al., 2013).
185 Males of A. cuneana not only transfer a large amount of substances during their first
186 copulation, they also shed virtually all their deciduous cornuti (genital spines) inside the first
187 female they mate with. Although experimental studies have not being conducted, deciduous
188 cornuti apparently cannot be “regenerated” as suggested by the observation in A. cuneana (this
189 study) and in other species with deciduous cornuti (Cordero & Miller, 2012; Anzaldo et al.,
190 2014) of mated males only with “scars” indicating the previous presence of cornuti, but never
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 191 with “incipient” spines suggesting a process of “re-growth” or “regeneration”. In virgin A.
192 cuneana males, cornuti form a "dense bundle" parallel to the longitudinal axis of the phallus,
193 occupying approximately one fifth of its length (personal observation; Phillips-Rodríguez &
194 Powell, 2007). These cornuti are relatively numerous (min–max: 19–34), elongated, slender,
195 spindle-shaped and pointed in both extremes (Fig. 1B). Their relative size and number suggests
196 that these structures could be costly to produce, and the fact that they can potentially damage the
197 genital tract of the female during and after copulation (Cordero, 2010; Cordero & Miller, 2012),
198 led us to suggest that they accomplish a reproductive function because otherwise selection would
199 have eliminated this trait (in fact, deciduous cornuti have disappeared several times in different
200 groups of Lepidoptera; Cordero & Miller, 2012; Anzaldo et al., 2014).
201 A. cuneana is considered a pest species (Phillips-Rodríguez & Powell, 2007; Juárez-
202 Gutiérrez et al., 2015) whose adult populations could reach high densities. If in these populations
203 adults emerge more or less synchronously and females tend to be monandrous, the time window
204 for mating would be small and males would have reduced mating opportunities which could
205 favour the large material investment observed in their first and probably only mating. If this
206 hypothesis is correct, we also predict that virgin males should have evolved to be prepared to
207 transfer a large ejaculate, which could favour the allocation of a large amount of resources to the
208 accessory glands. Due to the "necessity" to be ready to transfer an ejaculate, the diversion of
209 resources from reproductive tissues to somatic maintenance would not occur and this would
210 explain the lack of differences in longevity and mortality rate between mated and virgin males
211 (i.e. both types of males would have made a similar investment in ejaculate and cornuti
212 production, whether or not they mate). On the other hand, the fact that some males were able to
213 remate and transfer an ejaculate suggests that, sometimes, mated males are able to find receptive
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 214 females to copulate with. Our hypothesis suggests that studies should focus on measuring female
215 remating rates and male mating opportunities under field conditions.
216 Although we do not know the causes of the relatively high rate of mating failures of
217 virgin males (30.8% failed to mate despite being exposed to two different virgin females) and
218 previously mated males (7 out of 13 mated males exposed to a virgin female failed to remate),
219 one possible reason is mate choice. The high investment made by males in their first mating
220 could favour the evolution of male mate choice (Bonduriansky, 2001; Edward & Chapman,
221 2011). If this is the case in A. cuneana, mating “failures” of virgin males could result from males
222 rejecting females due to low quality; however this hypothesis is at odds with the idea that males
223 have reduced mating opportunities. On the other hand, in many Lepidoptera species ejaculates
224 contain nutritious and hormone-like substances that could increase female reproductive success
225 (Rutowski et al., 1987; Oberhauser, 1989; Royer & McNeil, 1993; Karlsson, 1998;Wiklund et
226 al., 1998; Torres-Vila & Jennions, 2005; South & Lewis, 2011). Thus, female choice could also
227 play a role especially (but not exclusively) in the case of males trying to remate given that they
228 transfer ejaculates whose mass is between 50% and 60% smaller than that transferred by virgin
229 males.
230 ACKNOWLEDGEMENTS
231 We thank Drs. Roxana Torres and Rogelio Macías for valuable suggestions, and Mirella Espino
232 and Raúl Martínez for technical help. The commentaries of three anonymous reviewers greatly
233 improved our manuscript. This research is part of the Doctoral thesis of Jaime César Camacho
234 García in the Posgrado en Ciencias Biológicas (Universidad Nacional Autónoma de México).
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314 expenditure in a polyandrous butterfly. Behavioral Ecology 9:20-25.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 316 Table 1 Descriptive statistics of experimental males and their material investment during
317 first copulations. WL: left wing length. Ejaculate weight is the weight of the bursa copulatrix
318 (ductus bursae + corpus bursae + accessory bursae) of mated females minus the average weight
319 of the bursa copulatrix of a sample of virgin females. #DC in CB: number of deciduous genital
320 spines (deciduous cornuti) shed by the male inside the corpus bursae of the female. Average ±
321 standard deviation, minimum value – maximum value, and sample size (n) are given in each cell.
Treatment WL (mm) Longevity (d) Ejaculate #DC in CB Male weight (mg) weight (mg) A. Survival Costs Experiment Virgin males 10.7 ± 0.55 19.6 ± 5.1 9.7–11.7 10–28 — — — n = 13 n = 16 Once-mated males 10.9 ± 0.63 18.5 ± 5.3 2.4 ± 0.62 26.8 ± 4.1 9.9–12.5 7–26 1.08–3.78 19–34 — n = 26 n = 26 n = 26 n = 25 Unsuccessful males 10.5 ± 0.75 15.8 ± 5.1 9.2–11.4 9–26 — — — n = 12 n = 12 Twice mated males 10.8 ± 0.59 20.2 ± 2.4 2.2 ± 0.33 24.3 ± 5.6 10–11.4 17–23 1.9–2.6 18–31 — n = 6 n = 5 n = 4 n = 4
B. Relative Investment Experiment Once-mated males 10.1 ± 0.65 1.84 ± 0.49 25.5 ± 3.9 20 ± 2.5 9.3–10.8 — 1.01–2.54 19–30 17.3–24.3 n = 8 n = 8 n = 8 n = 8 322
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: DB
Signum
Corpus bursae
323
324 Figure 1. The corpus bursa of female Amorbia cuneana (Tortricidae) moths and the
325 deciduous genital spines (cornuti) of males. (A) Corpus bursae of a recently mated female
326 filled with spermatophore and showing some deciduous cornuti; DB: ductus bursae. (B) A
327 deciduous cornutus extracted from the corpus bursa of a female.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: 328
329 Figure 2. Survival curves of male Amorbia cuneana moths (Tortricidae) from three mating
330 treatments. Males were kept virgin for life (solid line), mated once (dotted line) or did not mate
331 even though they were exposed twice to virgin females (dashed line). There were not significant
332 differences in survivorship between the three groups.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2598v1 | CC BY 4.0 Open Access | rec: 16 Nov 2016, publ: