Reproductive Behavior of Echinothrips americanus (Thysanoptera: Thripidae)

Reproductive Behavior of Echinothrips americanus (Thysanoptera: Thripidae) Most Thysanoptera possess a haplo-diploid reproductive mode and reproduce via arrhenotoky. Females can mature eggs successively throughout almost their entire life, but in most terebrantian thrips spermiogenesis is complete by adult male eclosion, and testes contain only mature spermatids. In parasitoid wasps this phenome- non of preadult spermiogenesis is described as prospermatogeny. It is unclear if prospermatogeny and this pre- determined sperm quantity have implications for mating strategy and fitness. In this study, we give a detailed description of mating behavior of the thripine species Echinothrips americanus, which largely corresponds with the only available data of another species of this family, Frankliniella occidentalis (Thysanoptera: Thripidae). With investigations using light microscopy, we describe for the first time the chronological sequence of internal processes during copulation. The release of male accessory gland material followed subsequently by spermato- zoa indicates production of a female-determined type 1 spermatophore. Despite prospermatogeny, males are able to inseminate 10 females with an equal amount of spermatozoa. It is only the quantity of glandular material that decreases with the number of previous copulations. Based on these new findings, the reproductive strategy of this species is discussed. Key words: sperm transfer, copulation, ejaculate volume, progenesis, prospermatogeny Detailed knowledge of reproduction is critical to managing those spe- Most Thysanoptera possess a haplo-diploid reproductive mode and cies that compete with us in the field, forest and greenhouse reproduce via arrhenotoky. Haploid males originate from unfertilized (Heming 1995). Each year, Thysanoptera cause millions of U.S. dol- eggs, whereas fertilized eggs develop into diploid females. Although fe- lars of crop loss (Lewis 1997a). By piercing and sucking the cells of males can mature eggs successively throughout almost their entire life, leaves, flowers, and fruits, thrips species, mainly from the suborder spermiogenesis in most terebrantian males is completed at adult eclo- Terebrantia, produce significant damage. Additionally, some of the sion (Bournier 1956, Heming 1970, Bode 1983). In parasitoid wasps, small species (1–2 mm) are able to transmit plant viruses this phenomenon of sperm progenesis completed prior to emergence is (Tospoviruses, Machlomovirus, Ilarvirus, Carmovirus, and described as prospermatogeny (Boivin et al. 2005). In contrast, synsper- Sobemovirus), bacteria, and fungi (Ullman et al. 1997, Gitaitis et al. matogenetic species are able to produce spermatozoa throughout their 2003, Jones 2005). whole lifetime. Whether prospermatogeny in Thysanoptera will result The natural process of thrips dispersal by wind has been supple- in sperm limitation and consequent implication for their mating strat- mented, as in many other insects, by human-mediated global trading egy, behavior, and fitness, is unclear. Detailed description of mating be- activities (Lewis 1997b). One example is the Poinsettia-Thrips, havior within the Terebrantia is available only for Frankliniella Echinothrips americanus, a species that is native to the eastern United occidentalis (Terry and Schneider 1993), F. schultzei (Milne et al. States (Stannard 1968). As a greenhouse pest, it has spread over 2007)and Scirtothrips aurantii (Rafter and Walter 2013). In F. occiden- Canada, Europe, Asia, and Northern Australia within the last 30 years talis, males attempt to mate with as many virgin females as possible. In (Vierbergen et al. 2006, Hoddle et al. 2012, Krueger et al. 2015). Its contrast, females reject mating attempts for many days after an initial broad host range and feeding damage (Oetting and Beshear 1993, copulation. Strong competition occurs among males to find virgin fe- Oetting et al. 1993, Vierbergen 1998, Trdan et al. 2003)makeita po- males (Terry and Schneider 1993). After a very brief precopulatory pe- tential major pest. But as in most species of Thysanopteraknowledge of riod, the male mounts a female, clasps her pterothorax with his legs reproduction is still anecdotal (Moritz 2006). and twists the tip of his abdomen underneath hers. Insertion of the V C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 17 July 2018 2 Journal of Insect Science, 2017, Vol. 17, No. 2 aedeagus follows. For successful copulation, antennal contact between Table 1. Observed mating behaviors with their description in E. americanus individuals, as well as stroking the females’ back with the mesothoracic legs of the male is necessary. Later, several periods appear, where no Behavior observed Description leg or head movement occur (Terry and Schneider 1993). On average, copulation lasts nearly 4 min (238.6 s, Terry and Schneider 1993). The Test start to male mounting Time between start of video record- ing and male mounting other reported species, F. schultzei and S. aurantii, show similar behav- Number of prior matings of male Number of previous matings of male ioral patterns (Milne et al. 2007, Rafter and Walter 2013)and similar before recorded copulation mating position. This is also known for several other Terebrantia Female rejects male prior mating Number of rejections of female prior (Franssen and Mantel 1964, Heming 1995) although complete knowl- copulation (same individuals) edge of the mating behavior is lacking. Initial contact to male mounting Time between initial contact (anten- Improving the knowledge of mating behavior and reproductive nal contact) and male mounting strategies was the aim of our study because understanding the biology Contact position Position of male and female during is essential for efficient pest control. Here, we give a precise description initial contact (head to head, head of mating behavior, mating frequency, duration, and chronological se- to abdomen, undetermined) quence of copulation. Additionally, we quantified the transfer of sper- Antennation Antennal contact of male and female Stroking Male palpate females back with his matozoa to get indications of possible effect of prospermatogeny. mesothoracic legs Duration of mating Time between insertion of aedeagus and separating Materials and Methods Calm period during mating Period during copulation without Animal Breeding and Keeping any observable movement of both sexes E. americanus laboratory culture was maintained at the University Female walking during mating Period of female walking with male of Halle, Germany. In acrylic cages (50  50  50 cm), they were on her back during copulation reared on Phaseolus vulgaris and Gossypium sp. as host plants. The culture was kept at 236 1 C, 606 10% RH with a photoperiod of 16:8 (L:D) h at 5000 Lux during the photophase. Leitz DMBRE (Leica) fitted with a Leica DFC 450C. Electron micro- To get virgin and naive males and females, females were allowed scopy samples were fixed, dehydrated, and embedded as samples for to lay eggs in wells of 12-well microtiter plates (Greiner, Cat.No: light microscopy. Specimens were cut into half by consecutive removing 665 180, Sigma-Aldrich, Munich, Germany). Each well (Ø 2.2 cm) of 5-mm sections (Leica SM 2000R microtome, Leica). Afterward, speci- was filled with 1.5 ml of 1.4% (w/v) agar and topped with a leaf mens were carefully washed in xylene and 90% EtOH to remove para- disc (Ø 1.6 cm) of P. vulgaris. Hatched larvae were reared separately plast. Hexamethyldisilazane was used to finish the samples. Later, these and sexed after eclosion. The plates were kept in a climate chamber were mounted on aluminium stubs with double-sided adhesive pads. under the same rearing conditions as the lab culture. Gold coating was performed with a Balzers SCD 004 sputter coater (Bingen, Germany) for 200 s at 20 mA. Samples were visualized with a Mating Behavior and Chronological Sequence scanning electron microscope Hitachi SEM S-2400 (Tokyo, Japan) at Virgin females of an age of 2–5 d post emergence, males with an age of 18 kV and documented on ILFORD FP 4 (Knutsford, Great Britain) roll either 1–3 d (later referred as “young males”) or 10–12 d (“old males”) film. Images of both microscopic techniques were adjusted for contrast were used to analyze mating behavior. Classification was adjusted after and brightness. Furthermore, males were restained on the scanned thereportedmeanlifespanof10–14d(Krueger et al. 2015). electron-microscopy images with the help of Photoshop CS5 (Adobe, Prepared wells of 12-well microtiter plate (same as described San Jose, CA). under rearing conditions) acted as observation arenas. Three females and one male were recorded for 1 h with a HD Webcam C525 Remating Frequency of Females (Logitech, Lausanne, Switzerland). Remating frequency was observed for copulations with the same To make our behavioral analysis comparable to other studies of male, as well as for copulations with new virgin males. For initial Terebrantia, we recorded the same behavioral traits as Terry and mating, couples were placed in an observation arena for 24 h. After Schneider (1993) (Table 1). Video playbacks have been used for initial mating, couples were separated. Every 24 h either the same investigation and analysis of duration and number of behaviors. male (as initial mating) (n ¼ 27) or a different male (n ¼ 18) was pre- Due to the small size of the insects, we were unable to analyze anten- sented again to each female. Within 30 min, number of mating nation and stroking via video playbacks. Therefore, data were sup- attempts and conducted matings were counted visually under a plemented by direct observations. Direct observations were stereo microscope (Leica S8 APO, Leica). After the 30-min test conducted in the same observation arenas under a stereomicroscope interval, the individuals were separated and kept in plates as (Leica S8 APO, Leica, Wetzlar, Germany). described before (animal breeding). The test runs until natural death Internal processes during mating and chronological sequences of individuals (females’ death in remating trial with different male, of copulation were analyzed by using histological methods. males’ death in remating trial with the same male). Couples were frozen in supercooled carnoy’s fluid (80 C) (abs. ethanol:chloroform:glacial acetic acid 6:3:1) at different time intervals after insertion of the phallus (1, 3, 5, 7 min, control: recently finished Quantification of Sperm Transfer copulation). Afterward, specimens were prepared for light or electron In order to quantify transfer of spermatozoa to the female and sperm microscopy. Specimens for light microscopy were dehydrated in a graded limitation in the males, one male and two females were placed in a ethanol series, embedded in Surgipath Paraplast (Leica, Prod. No. well of a 12-well Greiner-plate, prepared as described above and kept 39601006, Leica), cut into 5-mm sections (Leica SM 2000R microtome, in a climate chamber. Every 2 d, the male was transferred to two new Leica) and stained with hematoxylin-eosin. Slides were observed with a virgin females. The procedure was stopped after 0, 5, or 10 consecutive Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 3 matings of the male. To check for age-dependent effects, virgin males normal distribution using Shapiro–Wilk test (P> 0.05). Because of were treated similarly but without females. Afterward, all individuals data and residual distribution, data on mating behavior were were processed for histological analysis as illustrated in section analyzed by using nonparametric tests, whereas data on sperm Chronological Sequence. After DAPI staining (0.2 mg/ml Dapi in Aqua quantification were handled with parametric tests. dest., time:15 min,washing 3 Aqua dest.), samples were mounted in Anti-Fade-Medium (recipe after Jackson ImmunoResearch, www. jack Mating Behavior sonimmuno.com/technical/anti-fade.asp). Slides were examined with a Times of test start to mounting, initial contact to male mounting, light microscope Leitz DMBRE (Leica), fitted with a Leica DFC 450C, duration of mating, calm period, and walking of female during cop- and processed with ImageJ software (version 2.0.0, Wayne Rasband, ulation were analyzed with Mann–Whitney U test to distinguish http://imagej.nih.gov/ij/). Dapi intercalates with DNA; therefore, the between young and old males (P< 0.05). Frequency of contact posi- brightness of the coloration indicates the amount of DNA present (e.g., tion was analyzed with 2  3 Fishers exact test, P< 0.05. Darzynkiewicz 2010) and thus can be used to estimate spermatozoan quantity. Inverted 8-bit gray scale images (0¼ black, 255¼ white) were Frequency used to measure area, and mean gray values of the sperm ball within Frequency of mating attempts and mating in both remating trials the spermatheca or of the spermatozoa remaining within the testes (see were analyzed with 2  2 Fishers exact test, P< 0.05. Supp Appendices S1–S4 [online only]). To ensure comparable results, staining procedure, microscope setting, and room conditions were kept Sperm Quantification the same for all measurements. Further, mean gray value of testes or Area and index of gray values of testis and sperm ball within female sperm ball was taken in relation to background (minimal coloration were analysed with analysis of variance (ANOVA), means were sep- produced) and nucleus of somatic tissue (maximal coloration pro- arated by LSD post hoc test (P< 0.05). Data of both testes of one duced) to limit staining artefacts. individual showed no difference (paired t-test, area: t¼0.811, The index of the gray values of testes or sperm ball was calcu- P> 0.05; index of gray value: t¼0.336, P> 0.05) and were there- lated as follows: fore pooled. ðmean gray value of testes or sperm ball mean gray value backgroundÞ100 ðmean gray value nucleus  mean gray Results value backgroundÞ Mating Behavior and Chronological Sequence Mean gray value of background was measured in a 20  20 mm Copulation involves three phases: precopulation, copulation, and post- area outside of tissue. Maximal produced staining was measured on copulation (Table 2). The precopulatory phase starts with an initial nuclei of the gut (Supp Appendices S1–S4 [online only], measure- contact of both sexes. Generally, the male contacts the female with his ment of background not shown). antenna either in a head-to-head position (50%) or head to female abdomen/thorax position (27.7%). Then a receptive female lowers her abdomen and thorax, whereas the male mounts onto the female’s Statistical Analysis back. Unreceptive females throw off males by raising their abdomen. Data analysis was performed with WinStat for Excel (Fitch Males repeatedly attempt to mate with a female, even when she is not Software, Bad Krozingen, Germany) and SPSS Statistics 22 (IBM receptive. In the following copulation period, the male twists his abdo- Corp., Armonk, NY). Prior to analysis, data were checked for men underneath that of the female and tries to insert his aedeagus into Table 2. Mating behaviors of E. americanus analyzed from video observations, different letters indicate for significant differences Behavior n Duration in s 6 SD Frequency in % Range in s Precopulation Test start to male mounting Young males 10 633 6 533.8a 165–1641 Old males 9 1113.2 6 860.6a 105–2304 Initial contact to male mounting Young males 10 8 6 2.8a 5–13 Old males 9 9.7 6 5.1a 4–20 Contact position Head to head 18 50 Head to female abdomen 18 27.7 Undetermined 18 27.7 Copulation Duration Young males 10 403.2 6 31.28a 342–444 Old males 8 610.66 6 202.2b 389–995 Calm period Young males 10 297.3 6 128.2a 91–830 Old males 8 373.22 6 271.1a 88–423 Female walking Young males 10 11.1 6 35.1a 0–131 Old males 8 82.8 6 145.4a 0–111 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 4 Journal of Insect Science, 2017, Vol. 17, No. 2 the female genitalia. Meanwhile, he palpates her antennae with his (U-test, Z¼2.711, P ¼ 0.006), than older males. All other antennae, as well as stroking her back with his mesothoracic legs. recorded behaviors did not differ significantly. Successful insertion is followed by a calm period, which can vary During copulation, both parameres and the primitive aedeagus greatly in duration. This phase is characterized by no movement of any were completely inserted within the female s vagina (Fig. 1A–E). part of the body. Prior to separation of the couple, the female some- Endotheca and phallotheca were evaginated from the beginning of times starts to walk around with the male on her back (Table 2). copulation (Fig. 1D), and the paraphyses were located near the Mating behavior was analyzed to compare male age at mating. opening of the spermathecal duct (Fig. 1D and E). One minute after Younger males had a significantly shorter duration of mating insertion, the endotheca seems to be filled mainly with accessory Fig. 1. Copulation of E. americanus. (A–C) Male in blue color, SEM, light microscope, hemalaum-eosin stained (D–F). (A) Sagittal view of male and female in cop- ula, note in life male is on the back of the female, clasps her pterothorax with his legs and twists the tip of his abdomen underneath that of the female. (B) Male with genitalia fully inserted into female, cranio-dorsal view of couple. (C) Detailed view of inserted male genitalia (blue), note the parameres within the females’ vagina (big arrowhead). (D) Couple fixed 1 min after insertion of aedeagus, note the magenta colored male accessory gland secretion within endotheca (sagittal section). (E) Couple fixed 3 min after insertion of aedeagus, note the magenta colored male accessory gland secretion and few sperm (lilac) within endotheca (sagittal section). (F) Couple fixed 7 min after insertion of aedeagus, note the filled spermatheca of the female (sagittal section). cov, common oviduct; edth, endo- theca; hg, hindgut; o, oocyte; ovp, ovipositor; p, paramere; paed, primitive aedeagus; phtc, phallotheca; pph, paraphyses; sb, sperm ball; spth, spermatheca; spth d, spermathecal duct; vag, vagina. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 5 gland secretion (magenta-stained) and only few spermatozoa (Fig. 10-d-old virgin males were significantly smaller than in 5-d-old vir- 1D). After 3 min, more spermatozoa are visible within the endotheca gin males or in freshly emerged males (0-d virgin) (posthoc LSD, 10- (Fig. 1E). With increased time after the start of copulation, the num- d virgin vs. 5-d virgin, P ¼ 0.001; 10-d virgin vs. 0-d virgin, ber of visible spermatozoa within the endotheca increases. But sper- P< 0.001). Additionally, the area of sperm content decreased with matozoa were first observed in the spermatheca at 7 min after start the number of repeated copulations (Posthoc LSD, 5 mating vs. of copulation (Fig. 1F). After successful copulation, a sperm ball of 10 mating P< 0.001) (Fig. 2). twisted spermatozoa was located within the spermatheca (Fig. 1F). The index of gray value of sperm within the testes differs signifi- cantly between the conditions (ANOVA, df1 ¼ 4, df2 ¼ 74, F ¼ 6.211, P< 0.001). Age of the males had a significant effect on Remating Frequency of Females index of grey value. Freshly hatched adult males (0-d virgin) have In both conditions (remating with the same or a different male), significant lower index than 5-d virgin males (posthoc LSD, only one female mated again with the same male 3 d after the initial P ¼ 0.003). Index differ also between virgin and mated individuals copulation. But no significant differences in mating attempts or fre- (posthoc LSD, 5-d virgin vs. 5 mating, P ¼ 0.041; 10-d virgin vs. quency of mating were observed between both remating trials (mat- 10 mating P ¼ 0.001). Furthermore, the sperm number decrease ing attempts, Fishers exact test, P ¼ 0.766; matings, Fishers exact with consecutive matings (posthoc LSD, 5 mating vs. 10 mating test, P ¼ 1) (Table 3). P ¼ 0.03) (Fig. 3). The sperm ball size within the spermatheca decreased after copu- Quantification of Sperm Transfer lation with males with increased number of previous matings The area of sperm content within the testes differs between the con- (ANOVA, df1 ¼ 5, df2 ¼ 50, F ¼ 5.504, P ¼ 0.001) (Fig. 4). ditions (ANOVA, df1 ¼ 4, df2 ¼ 74, F ¼ 23.673, P< 0.001). We Interestingly, the index of gray value and therefore brightness of observed a decrease in sperm content over time, regardless of the sperm ball did not differ with number of previous matings by the number of previous matings: the area of sperm within the testes of male (ANOVA, df1 ¼ 5, df2 ¼ 50, F ¼ 1.239, P ¼ 0.306) (Table 4). Table 3. Frequency of mating attempts and matings in remating tri- als with different male and with the same male after initial copulation Discussion Mating Behavior Remating trial with Remating trial with Mating behavior was analyzed with video playbacks, as well as different male the same male direct observations. Behavior in E. americanus corresponded largely n (number of tested females) 18 27 to that of F. occidentalis described by Terry and Schneider (1993), F. Mating attempts 9 11 schultzei (Milne et al. 2007), and S. aurantii (Rafter and Walter Matings 0 1 2013). After the phase of initial contact, which was much shorter in Fig. 2. Area of sperm within testes of male E. americanus depending on age and number of previous matings. Solid line indicates the regression line, dashed line the 95% confidence interval. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 6 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 3. Box-plot of the index of grey value of sperm within testes of male E. americanus depending on age and number of previous matings calculated by the for- mula: Index ¼ (mean gray value of testes  mean gray value background)  100/(mean gray value nucleus  mean gray value background), bold line indicates the median, different letters indicate for significant differences. Fig. 4. Area of sperm ball within the spermatheca after a single mating by a female with males that had mated previously with other females in E. americanus. The number of previous matings by the male ranged from x1 to x10. Solid line indicates the regression line, dashed line the 95% confidence interval. E. americanus (about 8.61 s in E. americanus,19s in F. occidenta- males copulated significantly longer than younger males, a phenom- lis), mounting occurred. The male twisted his abdomen underneath enon known also in Tenebrio molitor (Carazo et al. 2011). that of the female and tried to insert his aedeagus into the female Presumably longer copulation is necessary to ensure transfer of genitalia. The duration of copulation varied with male age. Older enough sperm to the female, because we demonstrated a decrease in Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 7 Table 4. Index of gray value of sperm ball within spermatheca after a single mating by a female with males that had mated previously with other female E. americanus Number of previous matings by the male 1–2 3–4 5–6 7–8 9–10 N 15 6 14 8 13 Index of gray value 6 SD 95.59 6 8.7 111.50 6 11.9 97.0 6 22.37 93.02 6 24.15 98.69 6 13.04 The number of previous matings by the male ranged from  1to  10 spermatozoa quantity within the testes (see section below: sperm species only Tiarothrips subramanii is known to produce quantification), or older males are less effective in sperm transfer spermatophore-like objects (Ananthakrishnan 1990). (Carazo et al. 2011). However, copulation duration in E. americanus was much lon- Frequency ger than in F. occidentalis (Terry and Schneider 1993), but seems Remating frequency was extremely low in E. americanus. Although typical for terebrantian species (Caliothrips fasciatus 1–10 min, males mate multiple times, females reject mating after initial copula- Lewis 1973; Thrips major and Thrips fuscipennis, 6 min, Kirk 1985; tion, also noted by Li et al. (2014). Females of F. occidentalis refuse F. schultzei 9 min, Milne et al. 2007; S. aurantii, 13 min, Rafter and matings over a period of 15 d (Terry and Schneider 1993) and Walter 2013). Thrips tabaci is the only known species within the Terebrantia with repeated matings (Li et al. 2015). Frequent matings are also typical Chronological Sequence within the Tubulifera (Crespi 1986a,b, 1988a,b). Copulation starts with insertion of the phallus into the vagina. In E. americanus, a single copulation is enough to provide suffi- Phallotheca and endotheca are everted, presumably by hydrostatic cient sperm to fertilise eggs throughout female’s life time (Krueger pressure (Heming 1970, Pitkin 1972). The vagina is heavily et al. 2015). But monandry (female mate with one male during life- stretched, compared to virgin females. The placement of the gono- time) is rare in insects (Arnqvist and Nilsson 2000, Hosken et al. pore near the mouth of the spermathecal duct has also been 2009). described in F. fusca (Heming 1970). Sensilla on the outside of the parameres presumably assist in positioning the phallus within the female. Quantification of Sperm Transfer First, accessory gland material is transferred into the female, As expected, males show a significant decrease in area of spermato- whereas spermatozoa transfer occurs 3 min after phallus insertion. zoa within the testes in relation to the number of consecutive copu- Since no glandular material or spermatozoa were found inside the lations. Additionally, the lifespan of males also has an impact on vagina, and the paraphyses were located close to the spermathecal spermatozoa area quantity. Possibly muscle contraction in the testis duct, it can be assumed that the ejaculate migrates directly to the wall, suppression by other internal organs, or consumption of possi- duct and to the spermatheca. However, this process seems to take ble secretions within the testes are reasons for this observation. some time to complete, as filling of the spermatheca could be Brightness of sperm within testes, and therefore the amount of con- observed 7 min after insertion. After copulation, a sperm ball of tained sperm, decreases with the number of matings. But after 10 twisted spermatozoa is observable within the spermatheca. This copulations, the testes still contain 70% of spermatozoa compared sperm ball consists of a spherical capsule of densely packed secretion to 5-d-old virgin males. outside and less electron dense material and spermatozoa inside Freshly emerged virgin males had a lower index of gray value, (Teuber 2011). The encapsuled sperm ball is similar to that of other than 5- or 10-d-old virgin males. Possibly, maturation of sperm is species within the Thripidae (Heming 1995). not entirely completed at this point. Afterward (5- or 10-d-old mal- Bournier (1956) supposed this type of sperm ball to be a sperma- es), the age of virgin males had no impact on index of gray value of tophore. He uses the term spermatophore in a stricter sense as an spermatozoa within testes; therefore, no decrease in amount of sper- ampulla or capsule created by males and transferred to the female. matozoa with the age is detected. However, we did not test fertility Teuber (2011) argued that the unequal distribution of secretion of aged sperm, which is known to decrease with progressive age of within the sperm ball suggested a spermatophore rather than a coag- sperm cells (Reinhardt 2007). ulation within the female reproductive tract. But the gradual release In females, brightness of sperm ball within the spermatheca did of glandular secretion, and delayed transfer of spermatozoa shown not differ. In our study, females seem always to receive a similar in this study, might be reasons for the former reported distribution amount of spermatozoa, even from copulation with a tenfold mated patterns of spermatozoa and glandular material. Therefore, the male. But area of sperm ball significantly decreased with number of time-delayed transfer of secretion and spermatozoa, as well as the copulations. Probably the amount of transferred male accessory delivery near the spermathecal duct shown here, support Bode’s gland material decreases over time. In histological sections, flattened (1975) assumption of coagulation of the secretion within the and emptied accessory glands were conspicuous in 10 mated males spermatheca. (see Supp Appendices S5 and S6 [online only]). The sperm ball seems to be a special form of “female-determined Nonetheless, sperm limitation of males, prospermatogeny is of type 1 spermatophore” according to Gerber (1970), which is charac- minor importance. Males of E. americanus are able to successful terized by an ejection of secretion in a definite sequence before or inseminate up to 10 females. Males of Trichogramma evanescens after the spermatozoa and a form of coagulated material, deter- transfer relatively constant numbers of sperm to the first 10 females, mined by the female genital tract. This was already suspected by but subsequently fewer (Damiens and Boivin 2005). Transfer of Heming (1995). A similar “spermathecal spermatophore formation” accessory gland material might influence insemination success. Male is known only in the psocids Leptinotus patruelis (Finlayson 1949) Drosophila melanogaster run out of gland material after 4–5 consec- and Trogium pulsatorium (Klier 1956). Within the tubuliferan utive matings and fail to transfer sperm (Lefevre and Jonsson 1962). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 8 Journal of Insect Science, 2017, Vol. 17, No. 2 Crespi, B. J. 1988b. Risks and benefits from lethal male fighting in colonial A similar phenomenon is described in Drosophila silvestris after two Hoplothrips karnyi (Insecta: Thysanoptera). Behav. Ecol. Sociobiol. 22: matings (Schwartz 1991) and in bedbugs (Reinhardt et al. 2011). 293–301. Gland substances are linked not only to sperm transfer and sperm Damiens, D., and G. Boivin. 2005. Male reproductive strategy in protection but also to the induction of refractoriness in females, Trichogramma evanescens: sperm production and allocation to females. reduction of attractiveness, sperm competition, regulation of egg Physiol. Entomol. 30: 241–247. development, and induction of ovulation and oviposition (see Chen Darzynkiewicz, Z. 2011. Critical Aspects in Analysis of Cellular DNA 1984; Gillot 1988, 2003). Content. Curr. Protoc. Cytom. 56:7.2:7.2.1–7.2.8. doi.org/10.1002/ The different mating strategies of polygynous males and monan- 0471142956.cy0702s52. drous females within this species lead to a high risk of sexual compe- Finlayson, L. H. 1949. The life-history and anatomy of Leptinotarsus tition. It is not clear if females control the opportunity to mate with patruelis Pearman (Psocoptera-Atropidae). Proc. Zool. Soc. Lond. 119: 301–323. their limited receptivity, or if the monandry is male determined. In Franssen, C. J. H., and W. P. Mantel. 1964. Notes on copulation of thrips. addition to the resistance of the female to continuous male mating Tijdschrift Voor Entomol. 107: 341–347. attempts, males are able to manipulate female receptivity either by Gerber, G. H. 1970. Evolution of methods of spermatophore formation in their accessory gland products (see above) or by transferring of anti- pterygote insects. Can. Entomol. 102: 358–362. aphrodisiacs (Hosken et al. 2009). Such a pheromonal substance is Gillot, C. 1988. Accessory sex glands, pp. 356–471. In K.G. Adiyodi and R.D. detected for E. americanus (Krueger et al. 2016), but such substan- Adiyodi (eds.), Arthropoda-insecta: reproductive biology of invertebrates, ces have only a transient effect and serve as bridging until the female vol 3, Wiley, New York. is able to produce her own pheromone (Simmons 2001). Further Gillot, C. 2003. Male accessory gland secretions: Modulators of female repro- investigations are needed to distinguish the role of both sexes in this ductive physiology and behaviour. Annu. Rev. Entomol. 48: 163–184. mating strategy. Gitaitis, R. D., R. Walcott, M. L. Wells, J. C. Diaz Perez, and F. H. Sanders. 2003. Transmission of Pantoea ananatis, causal agent of center rot of onion, Nevertheless, prospermatogeny in E. americanus does not lead by tobacco thrips, Frankliniella fusca. Plant Dis. 87: 675–678. to a restriction of mating success and seems to be an adaptation to Heming, B. 1970. Postembryonic development of the male reproductive sys- their lifestyle. The short lifespan (10–14 d, Krueger et al. 2015), the tem in Frankliniella fusca (Thripidae) and Haplothrips verbasci brief opportunity to mate (low remating frequency of females), and (Phlaeothripidae) (Thysanoptera). Misc. Publ. Entomol. Soc. Am. 7: the small bodysize (because of the relation of energy costs for main- 237–272. taining gamete production and testes tissue in proportion to size) Heming, B. 1995. History of the Germ Line in Male and Female Thrips, pp. favors the development of prospermatogeny (see Boivin et al. 2005). 505–535. In B.L. Parker, M. Skinner, and T. Lewis (eds.), Thrips biology Therefore, as in many flower-living Thripidae, E. americanus seems and management. Plenum Press, New York. to be closely adapted to rapid reproduction and dispersal. Hoddle, M. S., A. Mound, and D. L. Paris. 2012. Thrips of California. CBIT Publishing, Queensland. Hosken, D. J., Stockley, T. Tregenza, and N. Wedell. 2009. Monogamy and Acknowledgments the Battle of the Sexes. Annu. Rev. Entomol. 54: 361–378. Jones, D. R. 2005. Plant viruses transmitted by thrips. Eur. J. Plant Pathol. We thank Christin Hesse for technical support. 113: 119–157. Kirk, W. 1985. Aggregation and mating of thrips in flowers of Calystegia sepium. Ecol. Entomol. 10: 433–440. Supplementary Data Klier, E. 1956. Zur Konstruktionsmorphologie des m€ annlichen Supplementary data are available at Journal of Insect Science online. Geschlechtsapparates der Psocopteren. Zoologische Jahrbu ¨ cher (Anatomie). 75: 207–286. Krueger, S., L. A. Mound, and G. B. Moritz. 2015. Offspring sex ratio and de- References Cited velopment are determined by copulation activity in Echinothrips ameri- canus MORGAN 1913 (Thysanoptera). J. Appl. Entomol. doi: 10.1111/ Ananthakrishnan, T. N. 1990. Reproductive biology of thrips. Indira jen.12280 Publishing House, Oak Park, MI. Krueger, S., G. B. Moritz, P. Lindemann, D. Radisch, and G. Tschuch. 2016. Arnqvist, G., and T. Nilsson. 2000. The evolution of polyandry: multiple mat- Male Phermones Influence the Mating Behavior of Echinothrips ameri- ing and female fitness in insects. Anim. Behav. 60: 145–164. canus. J. Chem. Ecol. 42: 294–299. Bode, W. 1975. Der Ovipositor und die weiblichen Geschlechtswege der € Lefevre, G., and U. B. Jonsson. 1962. Sperm transfer, storage and displace- Thripiden (Thysanoptera, Terebrantia). Z. Morphol. Okol. Tiere. 81: 1–53. ment, and utilization in Drosophila melanogaster. Genetics. 47: Bode, W. 1983. Spermienstruktur und Spermiohistogenese bei Thrips validus 1719–1736. Uzel (Insecta, Thysanoptera). Zool. Jahrbu ¨ cher (Anatomie). 109: 301–318. Lewis, T. 1973. Thrips: Their Biology, Ecology and Economic Importance. Boivin, G., S. Jacob, and D. Damiens. 2005. Spermatogeny as a life-history in- Academic Press London, New York, USA. dex in parasitoid wasps. Oecologia. 143: 198–202. Lewis, T. 1997a. Chemical control, pp. 567-93. In T. Lewis (ed.), Thrips as Bournier, A. 1956. Contribution a le ` tude de la parthe ` nogene ` se des Crop Pests. CAB International, Oxon and New York, USA. Thysanopteres et de sa cytology. Archiv. Zool. Exp. Ge ´ n. 93: 219–318. Lewis, T. 1997b. Flight and Dispersal, pp. 175-97. In T. Lewis (ed.), Thrips as Chen, P. S. 1984. The functional morphology and biochemistry of insect male Crop Pests. CAB International, Oxon and New York, USA. accessory glands and their secretions. Annu. Rev. Entomol. 29: 233–255. Li, X.-W.,-X. Jiang, X.-C. Zhang, A. M. Shelton, and J.-N. Feng. 2014. Post- Carazo, P., P. Molina-Vila, and E. 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Reproductive Behavior of Echinothrips americanus (Thysanoptera: Thripidae)

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© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.
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Abstract

Most Thysanoptera possess a haplo-diploid reproductive mode and reproduce via arrhenotoky. Females can mature eggs successively throughout almost their entire life, but in most terebrantian thrips spermiogenesis is complete by adult male eclosion, and testes contain only mature spermatids. In parasitoid wasps this phenome- non of preadult spermiogenesis is described as prospermatogeny. It is unclear if prospermatogeny and this pre- determined sperm quantity have implications for mating strategy and fitness. In this study, we give a detailed description of mating behavior of the thripine species Echinothrips americanus, which largely corresponds with the only available data of another species of this family, Frankliniella occidentalis (Thysanoptera: Thripidae). With investigations using light microscopy, we describe for the first time the chronological sequence of internal processes during copulation. The release of male accessory gland material followed subsequently by spermato- zoa indicates production of a female-determined type 1 spermatophore. Despite prospermatogeny, males are able to inseminate 10 females with an equal amount of spermatozoa. It is only the quantity of glandular material that decreases with the number of previous copulations. Based on these new findings, the reproductive strategy of this species is discussed. Key words: sperm transfer, copulation, ejaculate volume, progenesis, prospermatogeny Detailed knowledge of reproduction is critical to managing those spe- Most Thysanoptera possess a haplo-diploid reproductive mode and cies that compete with us in the field, forest and greenhouse reproduce via arrhenotoky. Haploid males originate from unfertilized (Heming 1995). Each year, Thysanoptera cause millions of U.S. dol- eggs, whereas fertilized eggs develop into diploid females. Although fe- lars of crop loss (Lewis 1997a). By piercing and sucking the cells of males can mature eggs successively throughout almost their entire life, leaves, flowers, and fruits, thrips species, mainly from the suborder spermiogenesis in most terebrantian males is completed at adult eclo- Terebrantia, produce significant damage. Additionally, some of the sion (Bournier 1956, Heming 1970, Bode 1983). In parasitoid wasps, small species (1–2 mm) are able to transmit plant viruses this phenomenon of sperm progenesis completed prior to emergence is (Tospoviruses, Machlomovirus, Ilarvirus, Carmovirus, and described as prospermatogeny (Boivin et al. 2005). In contrast, synsper- Sobemovirus), bacteria, and fungi (Ullman et al. 1997, Gitaitis et al. matogenetic species are able to produce spermatozoa throughout their 2003, Jones 2005). whole lifetime. Whether prospermatogeny in Thysanoptera will result The natural process of thrips dispersal by wind has been supple- in sperm limitation and consequent implication for their mating strat- mented, as in many other insects, by human-mediated global trading egy, behavior, and fitness, is unclear. Detailed description of mating be- activities (Lewis 1997b). One example is the Poinsettia-Thrips, havior within the Terebrantia is available only for Frankliniella Echinothrips americanus, a species that is native to the eastern United occidentalis (Terry and Schneider 1993), F. schultzei (Milne et al. States (Stannard 1968). As a greenhouse pest, it has spread over 2007)and Scirtothrips aurantii (Rafter and Walter 2013). In F. occiden- Canada, Europe, Asia, and Northern Australia within the last 30 years talis, males attempt to mate with as many virgin females as possible. In (Vierbergen et al. 2006, Hoddle et al. 2012, Krueger et al. 2015). Its contrast, females reject mating attempts for many days after an initial broad host range and feeding damage (Oetting and Beshear 1993, copulation. Strong competition occurs among males to find virgin fe- Oetting et al. 1993, Vierbergen 1998, Trdan et al. 2003)makeita po- males (Terry and Schneider 1993). After a very brief precopulatory pe- tential major pest. But as in most species of Thysanopteraknowledge of riod, the male mounts a female, clasps her pterothorax with his legs reproduction is still anecdotal (Moritz 2006). and twists the tip of his abdomen underneath hers. Insertion of the V C The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 journals.permissions@oup.com by Ed 'DeepDyve' Gillespie user on 17 July 2018 2 Journal of Insect Science, 2017, Vol. 17, No. 2 aedeagus follows. For successful copulation, antennal contact between Table 1. Observed mating behaviors with their description in E. americanus individuals, as well as stroking the females’ back with the mesothoracic legs of the male is necessary. Later, several periods appear, where no Behavior observed Description leg or head movement occur (Terry and Schneider 1993). On average, copulation lasts nearly 4 min (238.6 s, Terry and Schneider 1993). The Test start to male mounting Time between start of video record- ing and male mounting other reported species, F. schultzei and S. aurantii, show similar behav- Number of prior matings of male Number of previous matings of male ioral patterns (Milne et al. 2007, Rafter and Walter 2013)and similar before recorded copulation mating position. This is also known for several other Terebrantia Female rejects male prior mating Number of rejections of female prior (Franssen and Mantel 1964, Heming 1995) although complete knowl- copulation (same individuals) edge of the mating behavior is lacking. Initial contact to male mounting Time between initial contact (anten- Improving the knowledge of mating behavior and reproductive nal contact) and male mounting strategies was the aim of our study because understanding the biology Contact position Position of male and female during is essential for efficient pest control. Here, we give a precise description initial contact (head to head, head of mating behavior, mating frequency, duration, and chronological se- to abdomen, undetermined) quence of copulation. Additionally, we quantified the transfer of sper- Antennation Antennal contact of male and female Stroking Male palpate females back with his matozoa to get indications of possible effect of prospermatogeny. mesothoracic legs Duration of mating Time between insertion of aedeagus and separating Materials and Methods Calm period during mating Period during copulation without Animal Breeding and Keeping any observable movement of both sexes E. americanus laboratory culture was maintained at the University Female walking during mating Period of female walking with male of Halle, Germany. In acrylic cages (50  50  50 cm), they were on her back during copulation reared on Phaseolus vulgaris and Gossypium sp. as host plants. The culture was kept at 236 1 C, 606 10% RH with a photoperiod of 16:8 (L:D) h at 5000 Lux during the photophase. Leitz DMBRE (Leica) fitted with a Leica DFC 450C. Electron micro- To get virgin and naive males and females, females were allowed scopy samples were fixed, dehydrated, and embedded as samples for to lay eggs in wells of 12-well microtiter plates (Greiner, Cat.No: light microscopy. Specimens were cut into half by consecutive removing 665 180, Sigma-Aldrich, Munich, Germany). Each well (Ø 2.2 cm) of 5-mm sections (Leica SM 2000R microtome, Leica). Afterward, speci- was filled with 1.5 ml of 1.4% (w/v) agar and topped with a leaf mens were carefully washed in xylene and 90% EtOH to remove para- disc (Ø 1.6 cm) of P. vulgaris. Hatched larvae were reared separately plast. Hexamethyldisilazane was used to finish the samples. Later, these and sexed after eclosion. The plates were kept in a climate chamber were mounted on aluminium stubs with double-sided adhesive pads. under the same rearing conditions as the lab culture. Gold coating was performed with a Balzers SCD 004 sputter coater (Bingen, Germany) for 200 s at 20 mA. Samples were visualized with a Mating Behavior and Chronological Sequence scanning electron microscope Hitachi SEM S-2400 (Tokyo, Japan) at Virgin females of an age of 2–5 d post emergence, males with an age of 18 kV and documented on ILFORD FP 4 (Knutsford, Great Britain) roll either 1–3 d (later referred as “young males”) or 10–12 d (“old males”) film. Images of both microscopic techniques were adjusted for contrast were used to analyze mating behavior. Classification was adjusted after and brightness. Furthermore, males were restained on the scanned thereportedmeanlifespanof10–14d(Krueger et al. 2015). electron-microscopy images with the help of Photoshop CS5 (Adobe, Prepared wells of 12-well microtiter plate (same as described San Jose, CA). under rearing conditions) acted as observation arenas. Three females and one male were recorded for 1 h with a HD Webcam C525 Remating Frequency of Females (Logitech, Lausanne, Switzerland). Remating frequency was observed for copulations with the same To make our behavioral analysis comparable to other studies of male, as well as for copulations with new virgin males. For initial Terebrantia, we recorded the same behavioral traits as Terry and mating, couples were placed in an observation arena for 24 h. After Schneider (1993) (Table 1). Video playbacks have been used for initial mating, couples were separated. Every 24 h either the same investigation and analysis of duration and number of behaviors. male (as initial mating) (n ¼ 27) or a different male (n ¼ 18) was pre- Due to the small size of the insects, we were unable to analyze anten- sented again to each female. Within 30 min, number of mating nation and stroking via video playbacks. Therefore, data were sup- attempts and conducted matings were counted visually under a plemented by direct observations. Direct observations were stereo microscope (Leica S8 APO, Leica). After the 30-min test conducted in the same observation arenas under a stereomicroscope interval, the individuals were separated and kept in plates as (Leica S8 APO, Leica, Wetzlar, Germany). described before (animal breeding). The test runs until natural death Internal processes during mating and chronological sequences of individuals (females’ death in remating trial with different male, of copulation were analyzed by using histological methods. males’ death in remating trial with the same male). Couples were frozen in supercooled carnoy’s fluid (80 C) (abs. ethanol:chloroform:glacial acetic acid 6:3:1) at different time intervals after insertion of the phallus (1, 3, 5, 7 min, control: recently finished Quantification of Sperm Transfer copulation). Afterward, specimens were prepared for light or electron In order to quantify transfer of spermatozoa to the female and sperm microscopy. Specimens for light microscopy were dehydrated in a graded limitation in the males, one male and two females were placed in a ethanol series, embedded in Surgipath Paraplast (Leica, Prod. No. well of a 12-well Greiner-plate, prepared as described above and kept 39601006, Leica), cut into 5-mm sections (Leica SM 2000R microtome, in a climate chamber. Every 2 d, the male was transferred to two new Leica) and stained with hematoxylin-eosin. Slides were observed with a virgin females. The procedure was stopped after 0, 5, or 10 consecutive Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 3 matings of the male. To check for age-dependent effects, virgin males normal distribution using Shapiro–Wilk test (P> 0.05). Because of were treated similarly but without females. Afterward, all individuals data and residual distribution, data on mating behavior were were processed for histological analysis as illustrated in section analyzed by using nonparametric tests, whereas data on sperm Chronological Sequence. After DAPI staining (0.2 mg/ml Dapi in Aqua quantification were handled with parametric tests. dest., time:15 min,washing 3 Aqua dest.), samples were mounted in Anti-Fade-Medium (recipe after Jackson ImmunoResearch, www. jack Mating Behavior sonimmuno.com/technical/anti-fade.asp). Slides were examined with a Times of test start to mounting, initial contact to male mounting, light microscope Leitz DMBRE (Leica), fitted with a Leica DFC 450C, duration of mating, calm period, and walking of female during cop- and processed with ImageJ software (version 2.0.0, Wayne Rasband, ulation were analyzed with Mann–Whitney U test to distinguish http://imagej.nih.gov/ij/). Dapi intercalates with DNA; therefore, the between young and old males (P< 0.05). Frequency of contact posi- brightness of the coloration indicates the amount of DNA present (e.g., tion was analyzed with 2  3 Fishers exact test, P< 0.05. Darzynkiewicz 2010) and thus can be used to estimate spermatozoan quantity. Inverted 8-bit gray scale images (0¼ black, 255¼ white) were Frequency used to measure area, and mean gray values of the sperm ball within Frequency of mating attempts and mating in both remating trials the spermatheca or of the spermatozoa remaining within the testes (see were analyzed with 2  2 Fishers exact test, P< 0.05. Supp Appendices S1–S4 [online only]). To ensure comparable results, staining procedure, microscope setting, and room conditions were kept Sperm Quantification the same for all measurements. Further, mean gray value of testes or Area and index of gray values of testis and sperm ball within female sperm ball was taken in relation to background (minimal coloration were analysed with analysis of variance (ANOVA), means were sep- produced) and nucleus of somatic tissue (maximal coloration pro- arated by LSD post hoc test (P< 0.05). Data of both testes of one duced) to limit staining artefacts. individual showed no difference (paired t-test, area: t¼0.811, The index of the gray values of testes or sperm ball was calcu- P> 0.05; index of gray value: t¼0.336, P> 0.05) and were there- lated as follows: fore pooled. ðmean gray value of testes or sperm ball mean gray value backgroundÞ100 ðmean gray value nucleus  mean gray Results value backgroundÞ Mating Behavior and Chronological Sequence Mean gray value of background was measured in a 20  20 mm Copulation involves three phases: precopulation, copulation, and post- area outside of tissue. Maximal produced staining was measured on copulation (Table 2). The precopulatory phase starts with an initial nuclei of the gut (Supp Appendices S1–S4 [online only], measure- contact of both sexes. Generally, the male contacts the female with his ment of background not shown). antenna either in a head-to-head position (50%) or head to female abdomen/thorax position (27.7%). Then a receptive female lowers her abdomen and thorax, whereas the male mounts onto the female’s Statistical Analysis back. Unreceptive females throw off males by raising their abdomen. Data analysis was performed with WinStat for Excel (Fitch Males repeatedly attempt to mate with a female, even when she is not Software, Bad Krozingen, Germany) and SPSS Statistics 22 (IBM receptive. In the following copulation period, the male twists his abdo- Corp., Armonk, NY). Prior to analysis, data were checked for men underneath that of the female and tries to insert his aedeagus into Table 2. Mating behaviors of E. americanus analyzed from video observations, different letters indicate for significant differences Behavior n Duration in s 6 SD Frequency in % Range in s Precopulation Test start to male mounting Young males 10 633 6 533.8a 165–1641 Old males 9 1113.2 6 860.6a 105–2304 Initial contact to male mounting Young males 10 8 6 2.8a 5–13 Old males 9 9.7 6 5.1a 4–20 Contact position Head to head 18 50 Head to female abdomen 18 27.7 Undetermined 18 27.7 Copulation Duration Young males 10 403.2 6 31.28a 342–444 Old males 8 610.66 6 202.2b 389–995 Calm period Young males 10 297.3 6 128.2a 91–830 Old males 8 373.22 6 271.1a 88–423 Female walking Young males 10 11.1 6 35.1a 0–131 Old males 8 82.8 6 145.4a 0–111 Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 4 Journal of Insect Science, 2017, Vol. 17, No. 2 the female genitalia. Meanwhile, he palpates her antennae with his (U-test, Z¼2.711, P ¼ 0.006), than older males. All other antennae, as well as stroking her back with his mesothoracic legs. recorded behaviors did not differ significantly. Successful insertion is followed by a calm period, which can vary During copulation, both parameres and the primitive aedeagus greatly in duration. This phase is characterized by no movement of any were completely inserted within the female s vagina (Fig. 1A–E). part of the body. Prior to separation of the couple, the female some- Endotheca and phallotheca were evaginated from the beginning of times starts to walk around with the male on her back (Table 2). copulation (Fig. 1D), and the paraphyses were located near the Mating behavior was analyzed to compare male age at mating. opening of the spermathecal duct (Fig. 1D and E). One minute after Younger males had a significantly shorter duration of mating insertion, the endotheca seems to be filled mainly with accessory Fig. 1. Copulation of E. americanus. (A–C) Male in blue color, SEM, light microscope, hemalaum-eosin stained (D–F). (A) Sagittal view of male and female in cop- ula, note in life male is on the back of the female, clasps her pterothorax with his legs and twists the tip of his abdomen underneath that of the female. (B) Male with genitalia fully inserted into female, cranio-dorsal view of couple. (C) Detailed view of inserted male genitalia (blue), note the parameres within the females’ vagina (big arrowhead). (D) Couple fixed 1 min after insertion of aedeagus, note the magenta colored male accessory gland secretion within endotheca (sagittal section). (E) Couple fixed 3 min after insertion of aedeagus, note the magenta colored male accessory gland secretion and few sperm (lilac) within endotheca (sagittal section). (F) Couple fixed 7 min after insertion of aedeagus, note the filled spermatheca of the female (sagittal section). cov, common oviduct; edth, endo- theca; hg, hindgut; o, oocyte; ovp, ovipositor; p, paramere; paed, primitive aedeagus; phtc, phallotheca; pph, paraphyses; sb, sperm ball; spth, spermatheca; spth d, spermathecal duct; vag, vagina. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 5 gland secretion (magenta-stained) and only few spermatozoa (Fig. 10-d-old virgin males were significantly smaller than in 5-d-old vir- 1D). After 3 min, more spermatozoa are visible within the endotheca gin males or in freshly emerged males (0-d virgin) (posthoc LSD, 10- (Fig. 1E). With increased time after the start of copulation, the num- d virgin vs. 5-d virgin, P ¼ 0.001; 10-d virgin vs. 0-d virgin, ber of visible spermatozoa within the endotheca increases. But sper- P< 0.001). Additionally, the area of sperm content decreased with matozoa were first observed in the spermatheca at 7 min after start the number of repeated copulations (Posthoc LSD, 5 mating vs. of copulation (Fig. 1F). After successful copulation, a sperm ball of 10 mating P< 0.001) (Fig. 2). twisted spermatozoa was located within the spermatheca (Fig. 1F). The index of gray value of sperm within the testes differs signifi- cantly between the conditions (ANOVA, df1 ¼ 4, df2 ¼ 74, F ¼ 6.211, P< 0.001). Age of the males had a significant effect on Remating Frequency of Females index of grey value. Freshly hatched adult males (0-d virgin) have In both conditions (remating with the same or a different male), significant lower index than 5-d virgin males (posthoc LSD, only one female mated again with the same male 3 d after the initial P ¼ 0.003). Index differ also between virgin and mated individuals copulation. But no significant differences in mating attempts or fre- (posthoc LSD, 5-d virgin vs. 5 mating, P ¼ 0.041; 10-d virgin vs. quency of mating were observed between both remating trials (mat- 10 mating P ¼ 0.001). Furthermore, the sperm number decrease ing attempts, Fishers exact test, P ¼ 0.766; matings, Fishers exact with consecutive matings (posthoc LSD, 5 mating vs. 10 mating test, P ¼ 1) (Table 3). P ¼ 0.03) (Fig. 3). The sperm ball size within the spermatheca decreased after copu- Quantification of Sperm Transfer lation with males with increased number of previous matings The area of sperm content within the testes differs between the con- (ANOVA, df1 ¼ 5, df2 ¼ 50, F ¼ 5.504, P ¼ 0.001) (Fig. 4). ditions (ANOVA, df1 ¼ 4, df2 ¼ 74, F ¼ 23.673, P< 0.001). We Interestingly, the index of gray value and therefore brightness of observed a decrease in sperm content over time, regardless of the sperm ball did not differ with number of previous matings by the number of previous matings: the area of sperm within the testes of male (ANOVA, df1 ¼ 5, df2 ¼ 50, F ¼ 1.239, P ¼ 0.306) (Table 4). Table 3. Frequency of mating attempts and matings in remating tri- als with different male and with the same male after initial copulation Discussion Mating Behavior Remating trial with Remating trial with Mating behavior was analyzed with video playbacks, as well as different male the same male direct observations. Behavior in E. americanus corresponded largely n (number of tested females) 18 27 to that of F. occidentalis described by Terry and Schneider (1993), F. Mating attempts 9 11 schultzei (Milne et al. 2007), and S. aurantii (Rafter and Walter Matings 0 1 2013). After the phase of initial contact, which was much shorter in Fig. 2. Area of sperm within testes of male E. americanus depending on age and number of previous matings. Solid line indicates the regression line, dashed line the 95% confidence interval. Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 6 Journal of Insect Science, 2017, Vol. 17, No. 2 Fig. 3. Box-plot of the index of grey value of sperm within testes of male E. americanus depending on age and number of previous matings calculated by the for- mula: Index ¼ (mean gray value of testes  mean gray value background)  100/(mean gray value nucleus  mean gray value background), bold line indicates the median, different letters indicate for significant differences. Fig. 4. Area of sperm ball within the spermatheca after a single mating by a female with males that had mated previously with other females in E. americanus. The number of previous matings by the male ranged from x1 to x10. Solid line indicates the regression line, dashed line the 95% confidence interval. E. americanus (about 8.61 s in E. americanus,19s in F. occidenta- males copulated significantly longer than younger males, a phenom- lis), mounting occurred. The male twisted his abdomen underneath enon known also in Tenebrio molitor (Carazo et al. 2011). that of the female and tried to insert his aedeagus into the female Presumably longer copulation is necessary to ensure transfer of genitalia. The duration of copulation varied with male age. Older enough sperm to the female, because we demonstrated a decrease in Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 Journal of Insect Science, 2017, Vol. 17, No. 2 7 Table 4. Index of gray value of sperm ball within spermatheca after a single mating by a female with males that had mated previously with other female E. americanus Number of previous matings by the male 1–2 3–4 5–6 7–8 9–10 N 15 6 14 8 13 Index of gray value 6 SD 95.59 6 8.7 111.50 6 11.9 97.0 6 22.37 93.02 6 24.15 98.69 6 13.04 The number of previous matings by the male ranged from  1to  10 spermatozoa quantity within the testes (see section below: sperm species only Tiarothrips subramanii is known to produce quantification), or older males are less effective in sperm transfer spermatophore-like objects (Ananthakrishnan 1990). (Carazo et al. 2011). However, copulation duration in E. americanus was much lon- Frequency ger than in F. occidentalis (Terry and Schneider 1993), but seems Remating frequency was extremely low in E. americanus. Although typical for terebrantian species (Caliothrips fasciatus 1–10 min, males mate multiple times, females reject mating after initial copula- Lewis 1973; Thrips major and Thrips fuscipennis, 6 min, Kirk 1985; tion, also noted by Li et al. (2014). Females of F. occidentalis refuse F. schultzei 9 min, Milne et al. 2007; S. aurantii, 13 min, Rafter and matings over a period of 15 d (Terry and Schneider 1993) and Walter 2013). Thrips tabaci is the only known species within the Terebrantia with repeated matings (Li et al. 2015). Frequent matings are also typical Chronological Sequence within the Tubulifera (Crespi 1986a,b, 1988a,b). Copulation starts with insertion of the phallus into the vagina. In E. americanus, a single copulation is enough to provide suffi- Phallotheca and endotheca are everted, presumably by hydrostatic cient sperm to fertilise eggs throughout female’s life time (Krueger pressure (Heming 1970, Pitkin 1972). The vagina is heavily et al. 2015). But monandry (female mate with one male during life- stretched, compared to virgin females. The placement of the gono- time) is rare in insects (Arnqvist and Nilsson 2000, Hosken et al. pore near the mouth of the spermathecal duct has also been 2009). described in F. fusca (Heming 1970). Sensilla on the outside of the parameres presumably assist in positioning the phallus within the female. Quantification of Sperm Transfer First, accessory gland material is transferred into the female, As expected, males show a significant decrease in area of spermato- whereas spermatozoa transfer occurs 3 min after phallus insertion. zoa within the testes in relation to the number of consecutive copu- Since no glandular material or spermatozoa were found inside the lations. Additionally, the lifespan of males also has an impact on vagina, and the paraphyses were located close to the spermathecal spermatozoa area quantity. Possibly muscle contraction in the testis duct, it can be assumed that the ejaculate migrates directly to the wall, suppression by other internal organs, or consumption of possi- duct and to the spermatheca. However, this process seems to take ble secretions within the testes are reasons for this observation. some time to complete, as filling of the spermatheca could be Brightness of sperm within testes, and therefore the amount of con- observed 7 min after insertion. After copulation, a sperm ball of tained sperm, decreases with the number of matings. But after 10 twisted spermatozoa is observable within the spermatheca. This copulations, the testes still contain 70% of spermatozoa compared sperm ball consists of a spherical capsule of densely packed secretion to 5-d-old virgin males. outside and less electron dense material and spermatozoa inside Freshly emerged virgin males had a lower index of gray value, (Teuber 2011). The encapsuled sperm ball is similar to that of other than 5- or 10-d-old virgin males. Possibly, maturation of sperm is species within the Thripidae (Heming 1995). not entirely completed at this point. Afterward (5- or 10-d-old mal- Bournier (1956) supposed this type of sperm ball to be a sperma- es), the age of virgin males had no impact on index of gray value of tophore. He uses the term spermatophore in a stricter sense as an spermatozoa within testes; therefore, no decrease in amount of sper- ampulla or capsule created by males and transferred to the female. matozoa with the age is detected. However, we did not test fertility Teuber (2011) argued that the unequal distribution of secretion of aged sperm, which is known to decrease with progressive age of within the sperm ball suggested a spermatophore rather than a coag- sperm cells (Reinhardt 2007). ulation within the female reproductive tract. But the gradual release In females, brightness of sperm ball within the spermatheca did of glandular secretion, and delayed transfer of spermatozoa shown not differ. In our study, females seem always to receive a similar in this study, might be reasons for the former reported distribution amount of spermatozoa, even from copulation with a tenfold mated patterns of spermatozoa and glandular material. Therefore, the male. But area of sperm ball significantly decreased with number of time-delayed transfer of secretion and spermatozoa, as well as the copulations. Probably the amount of transferred male accessory delivery near the spermathecal duct shown here, support Bode’s gland material decreases over time. In histological sections, flattened (1975) assumption of coagulation of the secretion within the and emptied accessory glands were conspicuous in 10 mated males spermatheca. (see Supp Appendices S5 and S6 [online only]). The sperm ball seems to be a special form of “female-determined Nonetheless, sperm limitation of males, prospermatogeny is of type 1 spermatophore” according to Gerber (1970), which is charac- minor importance. Males of E. americanus are able to successful terized by an ejection of secretion in a definite sequence before or inseminate up to 10 females. Males of Trichogramma evanescens after the spermatozoa and a form of coagulated material, deter- transfer relatively constant numbers of sperm to the first 10 females, mined by the female genital tract. This was already suspected by but subsequently fewer (Damiens and Boivin 2005). Transfer of Heming (1995). A similar “spermathecal spermatophore formation” accessory gland material might influence insemination success. Male is known only in the psocids Leptinotus patruelis (Finlayson 1949) Drosophila melanogaster run out of gland material after 4–5 consec- and Trogium pulsatorium (Klier 1956). Within the tubuliferan utive matings and fail to transfer sperm (Lefevre and Jonsson 1962). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/17/2/66/3775834 by Ed 'DeepDyve' Gillespie user on 17 July 2018 8 Journal of Insect Science, 2017, Vol. 17, No. 2 Crespi, B. J. 1988b. Risks and benefits from lethal male fighting in colonial A similar phenomenon is described in Drosophila silvestris after two Hoplothrips karnyi (Insecta: Thysanoptera). Behav. Ecol. Sociobiol. 22: matings (Schwartz 1991) and in bedbugs (Reinhardt et al. 2011). 293–301. Gland substances are linked not only to sperm transfer and sperm Damiens, D., and G. Boivin. 2005. Male reproductive strategy in protection but also to the induction of refractoriness in females, Trichogramma evanescens: sperm production and allocation to females. reduction of attractiveness, sperm competition, regulation of egg Physiol. Entomol. 30: 241–247. development, and induction of ovulation and oviposition (see Chen Darzynkiewicz, Z. 2011. Critical Aspects in Analysis of Cellular DNA 1984; Gillot 1988, 2003). Content. Curr. Protoc. Cytom. 56:7.2:7.2.1–7.2.8. doi.org/10.1002/ The different mating strategies of polygynous males and monan- 0471142956.cy0702s52. drous females within this species lead to a high risk of sexual compe- Finlayson, L. H. 1949. The life-history and anatomy of Leptinotarsus tition. It is not clear if females control the opportunity to mate with patruelis Pearman (Psocoptera-Atropidae). Proc. Zool. Soc. Lond. 119: 301–323. their limited receptivity, or if the monandry is male determined. In Franssen, C. J. H., and W. P. Mantel. 1964. Notes on copulation of thrips. addition to the resistance of the female to continuous male mating Tijdschrift Voor Entomol. 107: 341–347. attempts, males are able to manipulate female receptivity either by Gerber, G. H. 1970. Evolution of methods of spermatophore formation in their accessory gland products (see above) or by transferring of anti- pterygote insects. Can. Entomol. 102: 358–362. aphrodisiacs (Hosken et al. 2009). Such a pheromonal substance is Gillot, C. 1988. Accessory sex glands, pp. 356–471. In K.G. Adiyodi and R.D. detected for E. americanus (Krueger et al. 2016), but such substan- Adiyodi (eds.), Arthropoda-insecta: reproductive biology of invertebrates, ces have only a transient effect and serve as bridging until the female vol 3, Wiley, New York. is able to produce her own pheromone (Simmons 2001). Further Gillot, C. 2003. Male accessory gland secretions: Modulators of female repro- investigations are needed to distinguish the role of both sexes in this ductive physiology and behaviour. Annu. Rev. Entomol. 48: 163–184. mating strategy. Gitaitis, R. D., R. Walcott, M. L. Wells, J. C. Diaz Perez, and F. H. Sanders. 2003. Transmission of Pantoea ananatis, causal agent of center rot of onion, Nevertheless, prospermatogeny in E. americanus does not lead by tobacco thrips, Frankliniella fusca. Plant Dis. 87: 675–678. to a restriction of mating success and seems to be an adaptation to Heming, B. 1970. Postembryonic development of the male reproductive sys- their lifestyle. The short lifespan (10–14 d, Krueger et al. 2015), the tem in Frankliniella fusca (Thripidae) and Haplothrips verbasci brief opportunity to mate (low remating frequency of females), and (Phlaeothripidae) (Thysanoptera). Misc. Publ. Entomol. Soc. Am. 7: the small bodysize (because of the relation of energy costs for main- 237–272. taining gamete production and testes tissue in proportion to size) Heming, B. 1995. History of the Germ Line in Male and Female Thrips, pp. favors the development of prospermatogeny (see Boivin et al. 2005). 505–535. In B.L. Parker, M. Skinner, and T. Lewis (eds.), Thrips biology Therefore, as in many flower-living Thripidae, E. americanus seems and management. Plenum Press, New York. to be closely adapted to rapid reproduction and dispersal. Hoddle, M. S., A. Mound, and D. L. Paris. 2012. Thrips of California. CBIT Publishing, Queensland. Hosken, D. J., Stockley, T. Tregenza, and N. Wedell. 2009. Monogamy and Acknowledgments the Battle of the Sexes. Annu. Rev. Entomol. 54: 361–378. Jones, D. R. 2005. Plant viruses transmitted by thrips. Eur. J. Plant Pathol. We thank Christin Hesse for technical support. 113: 119–157. Kirk, W. 1985. Aggregation and mating of thrips in flowers of Calystegia sepium. Ecol. Entomol. 10: 433–440. Supplementary Data Klier, E. 1956. Zur Konstruktionsmorphologie des m€ annlichen Supplementary data are available at Journal of Insect Science online. Geschlechtsapparates der Psocopteren. Zoologische Jahrbu ¨ cher (Anatomie). 75: 207–286. Krueger, S., L. A. Mound, and G. B. Moritz. 2015. 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