Mating alters the rate of development of ovarioles in the ladybird , Propylea dissecta ( Coleoptera : Coccinellidae )

The infl uence of female mating status on ovarian development of the ladybird, Propylea dissecta (Mulsant) (Coleoptera: Coccinellidae), was investigated under laboratory conditions. We assessed the extent to which ovariole development was affected by mating and for that we initially created a base line by observing age specifi c ovariole development. Results show that the number of follicles in each ovariole increased with the age of both virgin and mated females up to the age of 3 days, thereafter, no increase in number of follicles was recorded. Ovariole width also increased with age in both virgin and mated females up to 4 days, thereafter, no increase in ovariole width was recorded. The ovariole width of mated females was signifi cantly greater than that of virgin females. Egg maturation and the egg load started to increase at the age of 8 days in virgin females. Thereafter, it increased with increase in female age. While in mated females, immature eggs were recorded in their ovarioles from the age of 1 to 2 days. In mated females, however, the increase in the number of mature eggs per ovariole and egg load started when they were 3 days old. Egg load continuously increased with increasing female age. * Corresponding author. INTRODUCTION Ovarian development, a highly dynamic process (see review by Papaj, 2000), has been investigated extensively in insects, such as, fruit fl ies (Begun et al., 2006; Findlay et al., 2008), beetles (Kawazu et al., 2011, 2012; Ryall et al., 2013) and ladybird beetles (Hodek & Ceryngier, 2000; Ceryngier et al., 2004; Hodek, 2011; Nedved & Honek, 2011; Choate & Lundgren, 2013). Oocytes develop within ovarioles, arising from stem cells in the terminal fi lament and form a follicle along with trophocytes or nurse cells (Heming, 2003). The nurse cells act as a source and transport mechanism for vitellogenesis; sequestering, synthesizing compounds and delivering them to the growing oocytes via specialized channels. In advanced stages, mature oocytes can often be seen within each ovariole (Buning, 1994). A fi ve-stage rating system has been proposed to describe ovarian development in insects based on the length of the terminal follicle, the number and shape of follicles developing in each ovariole and presence of yellow yolk in the terminal oocyte (Jarvis & Copland, 1996). The maturation of ovaries in females is normally correlated with age, until sexual maturity is reached (Adams, 2000; Brent, 2010). However, factors other than age, such as food consumption (Papaj, 2000), temperature (Atlihan Eur. J. Entomol. 113: 44–50, 2016 doi: 10.14411/eje.2016.005 ISSN (online): 1802-8829 http://www.eje.cz


INTRODUCTION
Ovarian development, a highly dynamic process (see review by Papaj, 2000), has been investigated extensively in insects, such as, fruit fl ies (Begun et al., 2006;Findlay et al., 2008), beetles (Kawazu et al., 2011(Kawazu et al., , 2012;;Ryall et al., 2013) and ladybird beetles (Hodek & Ceryngier, 2000;Ceryngier et al., 2004;Hodek, 2011;Nedved & Honek, 2011;Choate & Lundgren, 2013).Oocytes develop within ovarioles, arising from stem cells in the terminal fi lament and form a follicle along with trophocytes or nurse cells (Heming, 2003).The nurse cells act as a source and transport mechanism for vitellogenesis; sequestering, synthesizing compounds and delivering them to the growing oocytes via specialized channels.In advanced stages, mature oocytes can often be seen within each ovariole (Buning, 1994).A fi ve-stage rating system has been proposed to describe ovarian development in insects based on the length of the terminal follicle, the number and shape of follicles developing in each ovariole and presence of yellow yolk in the terminal oocyte (Jarvis & Copland, 1996).

Stock maintenance
Adults of P. dissecta (twenty fi ve pairs) were collected from aphid infested agricultural crops in fi elds surrounding Lucknow (26°50´N, 80°54´E), U.P., India.They were paired and kept in transparent plastic Petri dishes (9.0 × 2.0 cm) under standard laboratory conditions (27 ± 1°C; 65 ± 5% R.H.; 14L : 10D photoperiod) and provided with an ad libitum supply of Aphis craccivora Koch (Hemiptera: Aphididae) (infested stems of cowpea, Vigna unguiculata L., reared in a glasshouse at 28 ± 1°C, 65 ± 5% R.H.).The eggs laid by this ladybird were placed in an environmental test chamber (Yorco Super Deluxe, YSI-440, New Delhi, India) until they hatched.The larvae were reared in plastic beakers (15.0 × 12.0 cm; 5 larvae per beaker) and provided an abundant supply of A. craccivora infested on cowpea twigs, which was replenished daily.The newly emerged adults were isolated and placed individually in Petri dishes (size as above) and thereafter used for experimental purposes.Beetles collected in the fi eld were added from time to time to the laboratory culture in order to avoid inbreeding.
In ladybirds, little work has been done on ovariole development and the factors affecting it, including the role of AGPs.The few studies on ovariole development in ladybirds indicate that the number of ovarioles is species specifi c and dependent on the environment of developing larvae (see Hodek et al., 2011). Ferrer et al. (2008) report a negative effect of suboptimal larval diet on ovariole number in A. bipunctata but the opposite trend was recorded in similar experiments on H. axyridis and a different population of A. bipunctata (Ware et al., 2008).Starvation also does not result in Harmonia axyridis developing fewer ovarioles but in a reduction in the percentage of mature ovarioles (Osawa, 2005).
The evidence linking ovariole development with age is contradictory and little is known about the underlying mechanisms of ovariole development in ladybirds.We, therefore, chose to investigate the normal ovariole development with increase in age and effect of mating on this phenomenon in the polyphagous ladybird beetle, Propylea dissecta.P. dissecta is small and has three morphs: pale, intermediate and typical (the pale and typical being most common).The male and female can be easily differentiated on the basis of patches present on the head and pronotum mol).These females were carefully dissected and the ovaries along with oviduct and common oviduct were removed and placed on a slide in a few drops of saline.These were then observed under a stereoscopic binocular microscope (Magnus, Olympus India, Noida) at 16× magnifi cation.Number of follicles per ovariole, ovariole width, number of mature eggs per ovariole and egg load (total number of mature eggs in an ovary) were recorded (following Jarvis & Copland, 1996).

(B) Effect of mating on ovariole development
This study was designed to investigate the effect of mating on ovariole development in ladybirds.For this purpose, a newly emerged 0-day-old (6 h post emergence) female was placed with a 10-day-old sexually mature unmated male in a Petri dish (9.0 × 2.0 cm).Females took 19.22 ± 1.30 min to establish mating and mated for 3.42 ± 0.14 h.Females showed resistance to being mated by rolling in her abdomen, kicking with her legs and moving fast, but the males managed to establish genital contact in all cases.After completion of mating, the male was removed and the female was isolated along with abundant A. craccivora.The Petri dishes were placed in an environmental test chamber (abiotic conditions similar to stock) for 24 h.After 24 h (i.e. at the age of 1 day), the mated female was killed and carefully dissected (as above).Similarly, 1, 2, 3 and 4-day-old females were mated with 10-day-old sexually mature unmated males and killed 24 h post mating, i.e. at the ages of 2, 3, 4 and 5 days, respectively.Virgin females of the same age as that of the mated females (at time of dissection) were also dissected and their reproductive systems measured as a control.The same measurements were recorded as in section (A).The observations were made on ten females (n = 10) per treatment.

Statistical analysis
Data obtained in experiment (A) on the number of follicles in each ovariole, ovariole width, number of mature eggs per ovariole, and egg load recorded for females of different ages (0, 1, 2, 3, 4, 5, 6, 7, 8 and 9 days) were subjected to polynomial regression analysis.To determine the effects of mating (Experiment B) on ovariole maturation, the number of follicles in each ovariole, ovariole width, number of mature eggs per ovariole and egg load were analyzed using two-way ANOVA followed by post hoc Tukey's test with female age (1, 2, 3 and 4 days) and mating status (mated and virgin females) as independent factors.All analyses were carried out using MINITAB 16 statistical software (Minitab Inc., State College, Pennsylvania, USA).
The number of mature eggs per ovariole signifi cantly increased with female age (y = 0.007x 3 -0.088x 2 + 0.287x -0.238; R² = 0.601; P < 0.01; Fig. 1c).Between 1 to 7 days, only immature eggs were recorded in the ovarioles.Mature eggs were fi rst recorded on day 8 in virgin females, followed by a rapid increase with increasing female age (Fig. 2).

(B) Effect of mating on ovariole development
The results of two-way ANOVA revealed that the number of follicles in each ovariole was affected by both fe- male age and mating status, and there was no interaction between these two factors (Table 1).The number of follicles increased rapidly after mating and statistically significant differences were recorded between mated and virgin females at the age of three and four days (Table 1).In both virgin and mated females, the ovariole width increased with increase in female age, however, it was signifi cantly wider in mated females than virgin female at ages of 3, 4 and 5 days (Table 1).

DISCUSSION
We show for the fi rst time that mating accelerates ovarian development in ladybirds.In addition, this study demonstrates that an increase in age leads to an increase in various ovariole development parameters: the number of follicles, ovariole width, the number of mature eggs and egg load.However, mating signifi cantly hastened this process, so much so that the number of follicles in 3 day old mated females was signifi cantly different from that in similar aged virgin females.
In our study, mating accelerated the rate of ovariole development.This is the fi rst time that such an effect has been reported in ladybirds.Such dramatic acceleration may be due to the transfer of seminal fl uid proteins and other male chemicals during mating that facilitate ovariole development (Quimio & Walter, 2000;Gillott, 2003).Seminal fl uid proteins are known to have a role in infl uencing yolk allocation, or inactivating genes for faster metabolism in the eggs of multiply mated females (Carvalho et al., 2006).Mating in some insects is also known to be essential for a number of female physiological processes, including, the initiation of vitellogenesis (Jin & Gong, 2001;Uchida et al., 2003), follicle production and maturation, and speed of ovarian development (Brunt, 1971;Horton et al., 2005).Mating is known to stimulate oogenesis in several insect species by triggering a hormonal response, mobilization of reserves, or through providing nutritional material (Gillott & Friedel, 1977;Wheeler, 1996).Although, this is not always so, as in Nezara viridula (L.) (Heteroptera: Pentatomidae) and other species of stinkbug, where mating does not infl uence ovary maturation (Masner, 1966;Davey et al., 1986).In stinkbugs, mating appears to be a key for female oviposition, since virgin females do not lay eggs (Odhiambo & Arora, 1973;Horton et al., 2005).The effects of mating on egg development and oviposition appear, therefore, to be variable within insect orders and even within some genera.
Thus, the present study indicates that ovariole development is an age related process, which is accelerated by mating.Mating in ladybirds may stimulate ovariole maturation by the males transferring proteins in their seminal fl uid that act either: (i) directly on ovariole development or (ii) indirectly by affecting the genes which regulate vitellogenesis.Further studies are therefore required to help identify the specifi c factors involved in this process.

Fig. 1 .
Fig. 1.The number of follicles per ovariole (a), ovariole width (b), number of mature eggs per ovariole (c) and egg load (d), recorded for virgin females of different ages of P. dissecta.

TABLE 1 .
Effect of female age and mating status on ovarian development in P. dissecta.