Introduction
Adequate nutrition is an important factor affecting growth, development and reproduction across animal taxa (Gordon, Reference Gordon1984; Hansen and Santer, Reference Hansen and Santer1995; Hanife, Reference Hanife2006). Variation in food availability has critical consequences for individual fitness via its effect on development and reproduction. Natural populations of many taxa, including polychaetes (Qian, Reference Qian1994), copepods (Ebert et al., Reference Ebert, Yamolsky and Van Noordwijk1993), insects (Grill et al., Reference Grill, Moore and Brodie1997), gastropods (Cheung and Lam, Reference Cheung and Lam1999), sea urchins (Thompson, Reference Thompson1982), snakes (Andren and Nilson, Reference Andren and Nilson1983), birds (Hussell and Quinne, Reference Hussell and Quinney1987) and mammals (Duquette and Millar, Reference Duquette and Millar1995) show modifications in reproduction in response to the variable food supply.
Nutritional availability in early development usually has strong downstream effects on adult fitness. When immature stages experience good nutritional conditions, adults usually mature earlier and are larger (Day and Rowe, Reference Day and Rowe2002). Developmental durations and body size are strongly correlated with fitness in a range of species (Rowe and Ludwig, Reference Rowe and Ludwig1991; Honek, Reference Honek1993; Abrams and Rowe, Reference Abrams and Rowe1996; Nylin and Gotthard, Reference Nylin and Gotthard1998). Altogether, these data suggest a ‘silver spoon’ scenario, in which favourable juvenile growth conditions lead to higher adult fitness. In contrast, the environment-matching hypothesis posits that this silver spoon effect depends on a continuation of favourable conditions into the adult stage. Food limitations that occur during adult stage (1) reduce offspring production (Korpimäki and Wiehn, Reference Korpimäki and Wiehn1998), (2) increase longevity (Harrison et al., Reference Harrison, Archer and Astle1984; Piper et al., Reference Piper, Partridge, Raubenheimer and Simpson2011), (3) increase susceptibility to predators and pathogens (Appleby et al., Reference Appleby, Anwar and Petty1999), and (4) decrease maternal investment (Hutto, Reference Hutto1990). Carry-over effects of nutrition from immature developmental stages to adulthood stages influence reproduction, ageing, development rate, tolerance to stress, and immunity (Yuval et al., Reference Yuval, Kaspi, Shloush and Warburg1998; Tu and Tatar, Reference Tu and Tatar2003; Andersen et al., Reference Andersen, Kristensen, Loeschcke, Toft and Mayntz2010; Dmitriew and Rowe, Reference Dmitriew and Rowe2011; Jiménez-Cortés et al., Reference Jiménez-Cortés, Serrano-Meneses and Córdoba-Aguilar2012; Leftwich et al., Reference Leftwich, Nash, Friend and Chapman2017). Nutritional restriction during growth reduces progeny immunity, growth and offspring fitness (Hoi-Leitner et al., Reference Hoi-Leitner, Romero-Pujante, Hoi and Pavlova2001; Bonduriansky and Head, Reference Bonduriansky and Head2007; Triggs and Knell, Reference Triggs and Knell2012).
Food availability during mating and/or post-mating also exerts strong effects over offspring production, viability and fitness (Rossiter, Reference Rossiter1996; Mitchell and Read, Reference Mitchell and Read2005). Food abundance during reproduction can also alter the mechanism of maternal effects involved in transferring material resources to offspring during prenatal development (Boersma, Reference Boersma1995; Rossiter, Reference Rossiter1996) thereby providing a potential avenue by which nutritional effect on mothers can be transferred to subsequent generations.
Aphidophagous ladybird beetles are economically important owing to their potential as biological control agents (Hodek et al., Reference Hodek, Van Emden and Honek2012; Omkar and Pervez, Reference Omkar and Pervez2016). Developmental and reproductive traits in aphidophagous ladybirds are subjected to strong food variations owing to the ephemeral nature of aphid populations (Borges et al., Reference Borges, Soares, Magro and Hemptinne2011). Ladybird larvae on a limited food supply develop into smaller adults (Kaddou, Reference Kaddou1960; Smith, Reference Smith1965; Kawauchi, Reference Kawauchi1990; Ng Reference Ng, Polgar, Dixon and Hodek1991; Agarwala et al., Reference Agarwala, Bardhanroy, Yasuda and Takizawa2001). In female ladybirds, both size and reduced prey are correlated with reduced egg output (Hodek and Honěk, Reference Hodek, Honěk, Hodek and Honěk1996; Obrycki et al., Reference Obrycki, Giles and Ormord1998). Likely, severe curtailment of prey availability also leads to reduced lifespan (Skorupa et al., Reference Skorupa, Dervisefendic, Zwiener and Pletcher2008). Thus, we have investigated the effect of quantitative food variation during pre and post-emergence and post-mating stage. The study aims to reveal the effect of stage-specific food supply on mating performance and reproductive traits in Propylea dissecta (Mulsant). We hypothesize that the effects of food availability at the time of development will not be restricted to the immature stages but will also be carried over to the adult stages. We also hypothesize that the plasticity in response to variation in food regime may lead to food availability induced life-history trait evolution.
Materials and methods
Study species
Propylea dissecta is an aphidophagous ladybird beetle of Oriental origin and quite prevalent in the agricultural and horticultural landscapes of North India, on aphids, Aphis gossypii Glover and Aphis craccivora Koch (Omkar and Pervez, Reference Omkar and Pervez2000a). It is polymorphic with three morphs, viz. pale, intermediate and typical (Pervez, Reference Pervez2002). The sexes can be differentiated quite easily in P. dissecta owing to sex-specific characteristic black markings on the head and pronotum (Omkar and Pervez, Reference Omkar and Pervez2000b).
Collection and rearing conditions
Adults of mixed body sizes were collected from agricultural fields surrounding Lucknow, India (26°50′N, 80°54′E) and brought to the laboratory for rearing. Field collected adults were paired in transparent plastic Petri dishes (14.5 × 1.5 cm2, one pair per dish) and placed in Biochemical oxygen demand (BOD) incubators (Yorco Super Deluxe, YSI-440 New Delhi, India) at 27 ± 2°C, 65 ± 5% R.H. and 14L: 10D photoperiod). They were provided with ad libitum daily replenished supply of cowpea aphid, A. craccivora (reared on host plant Vigna unguiculata L. in a greenhouse maintained at 25 ± 2°C, 65 ± 5% R.H. and 14L: 10D photoperiod). The pairs were checked daily for oviposition. The eggs laid were collected every 24 hours and incubated under the above abiotic conditions until hatching. Larvae were reared individually until pupation to adult emergence in plastic Petri dishes (size as above) and provided with ad libitum aphids until 10-days-old. The adults were taken from the stock to perform further experiments.
Optimization of food
To evaluate the effect of food on mating success of adults, abundant and restricted food regimes were provided. Standardization of food quantity for each regime was done. Aphis craccivora was provided to each predatory stage from the first instar to adulthood, including mated females at the following densities: 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 mg per day. The development of larvae and fecundity of mated females was recorded. The analysis revealed that a minimum of 5 mg aphid per day was required for completion of larval development and initiation of oviposition of viable eggs, albeit in limited quantities in mated females. Developmental and reproductive responses at 30 and 40 mg of aphid were statistically insignificant, and insignificant amounts of aphid were left uneaten, thus these were categorized as optimal diet. Thus, we used 5 mg of cowpea aphid as Restricted (R) diet and 50 mg as Abundant (A) diet.
Experimental protocol
Egg batches were randomly selected from the stock. Newly hatched larvae (less than 2-hours old) were subjected to one of two different food regimes (abundant or restricted) until pupation. Newly emerged adults from each regime were further split into two post-emergence dietary groups, viz. abundant and restricted. Both sexes were kept on similar diets throughout the treatment. On day 10 post-emergence, adults were paired and observed for mating. Post-mating, females were isolated and placed on any one of two post-mating dietary groups abundant and restricted. Precopulatory parameters, viz. time of commencement (TCM; time from the instant of cohabitation to intromission of aedeagus), latent period (LP; time from intromission of aedeagus to first abdominal shaking), mating duration (MD; time from intromission until dismounting) and postcopulatory parameters (fecundity and per cent egg fertility) were recorded. Each treatment was performed under similar conditions as mentioned above and each setup was replicated (n) 12 times.
Statistical analysis
Data of dependent variables (time to commence mating, latent period, mating duration, fecundity and per cent egg fertility) were first tested for normality (Kolmogorov–Smirnov's test) and homogeneity of variances (Bartlett's test). On being found normally distributed, time to commence mating, latent period and mating duration were subjected to a generalized linear model with a pre-emergence and post-emergence food regime treated as independent factors. However, the data of fecundity and per cent egg fertility was also subjected to a generalized linear model involving three independent factors viz., pre-emergence, post-emergence and post-mating food regime. All statistical analyses were conducted using SPSS statistical software (version 20.0, SPSS Company, Chicago, USA).
Results
Time to commence mating was significantly influenced by both pre-emergence (W = 15.144, P < 0.0001, df = 1, 95) and post-emergence (W = 6.635, P = 0.010, df = 1, 95; Table 1) food regimes. Mating commenced early in pairs reared on abundant food supply at both life stages. Individuals with even one restricted regime took similar durations to commence mating regardless of the stage at which restricted prey was provided (fig. 1a).
Effect of pre- and post-emergence food regime on mating behaviour (a) time to commence mating (TCM), (b) latent period (LP), (c) mating duration (MD) in P. dissecta. Values are Mean ± SE. Small letters denote the comparison of means across all the treatments while capital letters denote within post-emergence food regime or across the same pre-emergence food regime respectively. Similar letters indicate a lack of significant difference between the mating pairs at P > 0.05. * In food regime treatments mentioned on X-axis the first letter indicates pre-emergence food regime (from egg hatching until pupation), while the second letter denotes post-emergence food regime (from adult emergence until adult maturity).
Showing the effect of pre- and post-emergence food regime on mating parameters and their interactions
PE, pre-emergence; Post-E, post-emergence; TCM, time to commence mating; LP, latent period; MD, mating duration.
Latent period was not significantly influenced by pre-emergence food regime (W = 1.841, P = 0.175, df = 1, 95), but it was significantly influenced by post-emergence food regime (W = 4.484, P = 0.035, df = 1, 95; Table 1). When reared in abundant pre-emergence regime, adults reared under abundant post-emergence regime had a shorter latent period than those with restricted ones. However, treatments with even one restricted regime had a similar latent period, regardless of the stage it was given at (fig. 1b).
Mating duration was affected significantly by both pre-emergence (W = 16.955, P < 0.0001, df = 1, 95) and post-emergence (W = 23.705, P < 0.0001, df = 1, 95) food regimes. The interaction amongst the two food regimes (W = 6.637, P < 0.0001, df = 1, 95; Table 1) also significantly affected mating duration. The longest mating duration was found in pairs reared on abundant food supply for their entire life. Within each pre-emergence regime, the restricted post-emergence regime was more limiting on mating duration (fig. 1c). Adults provided with both restricted and abundant food regimes, irrespective of the life stage they were supplied at, show similar mating durations.
Pre-emergence (W = 46.802, P < 0.0001, df = 1, 95), post-emergence (W = 58.695, P < 0.0001, df = 1, 95) and post-mating (W = 37.649, P < 0.0001, df = 1, 95) food regimes significantly affected fecundity. Interactions between pre-emergence and post-mating food regime (W = 9.075, P = 0.003, df = 1, 95; Table 2) were significant. Food supply during all three stages had a significant effect over fecundity. Individuals reared on restricted food regime at any of the two stages had reduced fecundity compared to those with just one restricted food supply (fig. 2a). The egg-laying pattern observed in these ladybirds indicates plasticity during food stress, as it may be physiologically costly for females to balance between reproduction and survival.
Effect of pre-, post-emergence and post-mating food regime on reproductive output (a) Fecundity (b) % fertility in P. dissecta. Values are Mean ± SE. Small letters denote the comparison of means across all the treatment respectively. Similar letters indicate a lack of significant difference between the mating pairs at P > 0.05. * In food regime treatments mentioned on X-axis the first letter indicates pre-emergence food regime (from egg hatching until pupation), while the second letter denotes post-emergence food regime (from adult emergence until adult maturity) and third letters indicate post-mating (food regime after mating).
The result of Generalized Linear Model with the factors pre-, post-emergence and post-mating food regime showing their effects on fecundity and % fertility
PE, pre-emergence; Post-E, post-emergence; PM, post-mating.
Per cent egg fertility was significantly influenced by pre-emergence (W = 27.093, P < 0.0001, df = 1, 95), post-emergence (W = 31.512, P < 0.0001, df = 1, 95) and post-mating (W = 21.752, P < 0.0001, df = 1, 95; Table 2) food regimes. Interaction between post-emergence and post-mating food regime was also significant (W = 3.882, P = 0.049, df = 1, 95). Individuals reared on abundant food supply had the highest egg fertility, while those reared on other treatments differed insignificantly (fig. 2b).
Discussion
Food availability at different life stages (pre and post-emergence and post-mating) differentially shapes behavioural and reproductive traits in adults. As expected, ladybirds reared on abundant food from pre-emergence through to post-mating were the fastest at establishing mating, mated for the longest duration and were the most fecund with the highest percentage egg fertility. Similarly, those reared on a restricted diet at all three stages fared the worst. However, in individuals where food supply was abundant before pupation, beetles may compensate for overall body size and mass, but body composition may still be affected (Scriber and Slansky, Reference Scriber and Slansky1981; Plaistow and Siva-Jothy, Reference Plaistow and Siva-jothy1999). The nutrition attained before emergence can be transferred to the adult stage; while food consumed during adult stages possibly directly affects physiology and reproduction.
Adults reared on the restricted food regime anytime during their life showed the delayed establishment of matings which may be due to: (1) poor male vitality, and/or (2) female avoidance of costly matings with poor quality males (Parker, Reference Parker1983; Gwynne, Reference Gwynne1993). Females probably use the male activity as an indicator of their fitness (Kotiaho et al., Reference Kotiaho, Alatalo, Mappes and Parri1996; Byers et al., Reference Byers, Hebets and Podos2010). The pairs with one restricted food regime at any two life stages took similar durations to establish mating. The latent period was influenced by post-emergence food regimes, which can contribute to their mating performance. Food shortage during the adult stage may result in a longer latent period owing to decreased vitality (Agarwala et al., Reference Agarwala, Yasuda and Sato2008).
The maximum mating duration was observed in individuals that had received abundant food their entire lives, while the opposite was observed in the case of those reared on restricted food. Past studies have also shown that well-fed individuals are at profit in several aspects when compared with those from food-limited environments, viz. higher probability of being selected as mates, better performance during competition, receiving a higher number of matings, longer copulation durations, better quantity and quality of sperms, larger quantities of ovipositional stimulants, accessory gland proteins and nuptial gifts to mates (Engqvist and Sauer, Reference Engqvist and Sauer2003; Hebets et al., Reference Hebets, Wesson and Shamble2008; Eraly et al., Reference Eraly, Hendrickx and Lens2009; Albo et al., Reference Albo, Toft and Bilde2012). Nutrition acquired at larval stage may result in stored energy reserves, such as fat and proteins, which influences reproductive fitness among adults via effects on both body size and the composition of the adult body (Boggs and Freeman, Reference Boggs and Freeman2005). However, restricted food during post-emergence stage was a higher limiting factor on mating duration in this study. This may indicate that the resources accumulated in the larval stages are utilized early in adult life when reared on restricted prey, probably for sustenance, and thereby less energy is left for investing in mating duration (Dixon, Reference Dixon2000). Previous studies have also shown that post-emergence food regime affects ejaculate size (Perry and Rowe, Reference Perry and Rowe2010), nuptial gifts (Cratsley et al., Reference Cratsley, Rooney and Lewis2003; Boggs, Reference Boggs2018), sexual activity (Engqvist and Sauer, Reference Engqvist and Sauer2003), and individuals' survival rate (Sibly and Hone, Reference Sibly and Hone2002; Davis et al., Reference Davis, Nager and Furness2005).
Reduced fecundity was observed in pairs reared on a restricted diet at any two life stages. However, individuals exposed to food restriction throughout pre-emergence resulted in lesser total egg production than individuals subjected to post-emergence feeding treatment. This may be attributed to (1) the non-availability of critical dietary nutrients which may be involved in facilitating oogenesis and development (Fox et al., Reference Fox, Martin, Thakar and Mousseau1996; Cope and Fox, Reference Cope and Fox2003), (2) resorption of eggs (Boggs and Ross, Reference Boggs and Ross1993), (3) reduction in ancillary fluid production, (4) downregulation of immune response at the time of reproduction (French, Johnston and Moore, Reference French, Johnston and Moore2007), (5) reduced longevity during food stress (Kaitala, Reference Kaitala1991), (6) reduction in the number of eggs (Dixon and Guo, Reference Dixon and Guo1993), and (7) reduction in egg size which in turn might limit female reproductive output (Droney, Reference Droney1996). Food shortage during immature stages is known to decrease growth rates as well as reproductive output later in life (Briegel, Reference Briegel1990; Berrigan and Charnov, Reference Berrigan and Charnov1994; Blanckenhorn, Reference Blanckenhorn1998; Fischer and Fiedler, Reference Fischer and Fiedler2001; Vargas et al., Reference Vargas, Michaud and Necholos2012). While food limitation at any level was detrimental, scarcity of food at the post-emergence stage especially reduced fecundity. This can be attributed to the diversion of resources to sustenance rather than reproduction. Such food restriction at different stages may yield different reproductive outputs.
This study shows that for maximum fertility of eggs, abundant food is necessary at all life stages. Previous studies also support the same findings; holometabolous insects can accumulate resources as larvae/juvenile stages that augment or supplement adult nutrient intake, which in turn increases the reproductive success of individuals in both sexes (De Block and Stoks, Reference De Block and Stoks2005; Pechenik, Reference Pechenik2006). Thus, higher fertility of eggs can be attributed to the good condition of adults. Adults reared in good conditions are assumed to invest their available resources abundantly in reproduction (Van Noordwijk and De Jong, Reference Van Noordwijk and De Jong1986). However, the interactions among all the food regimes showed insignificant effects over per cent egg fertility, which possibly suggests that male condition also could affect egg fertility through their ejaculates quality and quantity. Previous studies on ladybirds that differ in objective and design to our study suggest that males modify the egg viability by providing ejaculates, accessory nutrients, and nuptial gifts for fertilization (Mishra and Omkar, Reference Mishra and Omkar2004; Perry and Rowe, Reference Perry and Rowe2008; Bista and Omkar, Reference Bista and Omkar2013; Michaud et al., Reference Michaud, Bista, Mishra and Omkar2013; Singh et al., Reference Singh, Mishra and Omkar2016). Male age has also been shown to decrease ejaculate quality and quantity (Pervez et al., Reference Pervez, Omkar and Richmond2004; Srivastava and Omkar, Reference Srivastava and Omkar2004), together driving higher egg fertility. Moreover, it is likely that middle-aged individuals may perform better. Recent studies on arthropods have investigated the effect of male or female age on sperm quality and quantity (Hayashi, Reference Hayashi1999; Green, Reference Green2003), sperm transfer rates (Jones et al., Reference Jones, Featherston, Paris and Elgar2006), last male sperm precedence (LaMunyon, Reference Lamunyon2001; Schafer and Uhl, Reference Schafer and Uhl2002; Mack et al., Reference Mack, Priest and Promislow2003; Radwan et al., Reference Radwan, Michalczyk and Prokop2005), sperm competitive ability (Service and Fales, Reference Service and Fales1993; Jones et al., Reference Jones, Featherston, Paris and Elgar2006), the musculature of the genital tract (Mack et al., Reference Mack, Hammock and Promislow2002) and sperm storage (Taylor et al., Reference Taylor, Kaspi, Mossinson and Yuval2001).
To summarize, food conditions experienced during early life stages may result in developing adaptations for the future environment. However, this condition-dependent response is not limited to the larval stage, as adults are also under constant selection pressure. Thus, our study suggests that (i) food is of paramount importance and any change in food supply will affect individual development, (ii) food stress in early life exerts a strong effect on reproductive fitness which might result in small body size and reduced fecundity, (iii) mating behaviour was affected by restricted food regardless of whether it was in the preemergence or post-emergence period, and (iv) fecundity was affected by the number of abundant regimes, not the sequence. As long as the ladybird had ‘two square meals’ in any one of the three feeding stages, the stress of a solo bout of scarcity could be effectively dealt with. We eagerly await further studies that extend the general approaches taken here to understand more about the effects of stage-specific food stress on their life history pattern and their carry-over effects.
Acknowledgements
PS gratefully acknowledges António Onofre Soares, Auxiliar Professor at the Faculty of Sciences and Technology, University of the Azores, Portugal and Alicia Katie-Mae Hodson, Bristol, UK for improving the language of the paper. PS also acknowledges Department of Zoology, University of Lucknow, Lucknow, Uttar Pradesh, India for providing facilities for this research.
