- Email: [email protected]
Multigenerational maternal inhibition of prepupal diapause in two Trichogramma species (Hymenoptera: Trichogrammatidae)
Journal of Insect Physiology 81 (2015) 14–20
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Multigenerational maternal inhibition of prepupal diapause in two Trichogramma species (Hymenoptera: Trichogrammatidae) Sergey Ya. Reznik ⇑, Konstantin G. Samartsev Laboratory of Experimental Entomology, Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia
a r t i c l e
i n f o
Article history: Received 30 March 2015 Received in revised form 1 June 2015 Accepted 23 June 2015 Available online 24 June 2015 Keywords: Diapause Maternal effect Interval timer Photoperiod Trichogramma
a b s t r a c t It is known that in some insect species the incidence of diapause among the progeny of females that had undergone diapause is relatively low or zero even under strong diapause-inducing conditions. Moreover, the maternal inhibition, preventing the induction of a maladaptive diapause in spring, can persist over several generations. This multigenerational effect based on hypothetical ‘interval timer’ was thoroughly studied in Aphididae. We first described a similar phenomenon in Hymenoptera: laboratory experiments demonstrated that the proportion of diapausing progeny of Trichogramma females that had undergone diapause was practically zero independently of photoperiodic and temperature conditions used (day lengths of 12 and 18 h and temperatures of 12–15 °C). Then the ability to enter diapause recovered gradually and returned to the normal level over two (in Trichogramma telengai) or even five (in Trichogramma principium) generations. We conclude that the observed effect may be based on an interval timer similar to that in aphids. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Various maternal effects on diapause induction have been found in many insect species. Based on external manifestations, these effects can be separated into two categories: (1) maternal exposure to diapause-inducing environmental stimuli results in producing diapausing progeny and (2) females that had undergone diapause are incapable of producing diapausing progeny even under strong diapause-inducing conditions. Maternal effects of the first type were often detected when the progeny has no time to react to diapause-inducing stimuli (e.g., in insect species with an embryonic diapause) or cannot perceive environmental cues (e.g., in Diptera and Hymenoptera with larvae living in soil and other cryptic habitats). The reactions of the second type are most probably aimed at preventing the induction of an untimely maladaptive diapause in the progeny of females that had undergone diapause, e.g., when the first spring generation of a polyvoltine species is subjected to late frosts (Denlinger, 1998, 2002; Lees, 1966; Mousseau and Fox, 1998; Ogawa and Miura, 2014; Saunders et al., 2002; Saunders, 2010). In a broad sense, all the above maternal effects can be considered as transgenerational developmental plasticity, but their ⇑ Corresponding author at: Laboratory of Experimental Entomology, Zoological Institute, Russian Academy of Sciences, Universitetskaya nab., 1, St. Petersburg 199034, Russia. E-mail address: [email protected] (S.Ya. Reznik). http://dx.doi.org/10.1016/j.jinsphys.2015.06.012 0022-1910/Ó 2015 Elsevier Ltd. All rights reserved.
physiological mechanisms (in the rare cases when they were revealed) were shown to be very different. In the simplest cases, maternal effects on diapause are based on a direct transfer of nutrients from mother to progeny. For example, in Bombyx mori L. the diapause hormone of the female influences carbohydrate and polyol content of the egg and thus determines diapause of the embryo (Denlinger, 2002; Yamashita, 1996). Such ‘metabolic effects’ resulting from a transfer of nutrients directly influencing the development of progeny were mostly detected in the first generation. In some cases, such effects were found in eggs but never in larvae of the second generation because the nutrient was too much diluted (Hercus and Hoffmann, 2000; Kyneb and Toft, 2006). The ‘signal effects’ are based either on the transmission of cytoplasmic factors (RNA, hormones) or on the epigenetic inheritance in the strict sense of the term (transgenerational transmission of variations in DNA expression). It is well known that environmentally induced epigenetic changes ensuring transmission of phenotypic characters without modification to gene sequences can be often traced over several generations (Bossdorf et al., 2008; Ho and Burggren, 2010; Jablonka and Raz, 2009; Uller, 2008). Of course, metabolic and signal mechanisms are not mutually exclusive, but long-term effects are most probably based on transfer of signals rather than nutrients. In the regulation of insect diapause, these effects are often manifested as ‘interval timers’ which measure time intervals independently of the number of elapsed generations, although ‘generation counters’ reducing the effect in accordance
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
with the number of generations passed can be also conceived (Brodel and Schaefers, 1979; Campbell and Tregidga, 2006; Denlinger, 1998, 2002; Dixon, 1971, 1972; Lees, 1960, 1966; Lushai et al., 1996; Margaritopoulos et al., 2002; Ogawa and Miura, 2014; Saunders et al., 2002). At present, biochemical and molecular mechanisms of diapause induction are intensively investigated (Denlinger, 2002; Denlinger et al., 2012) but ‘classic physiological experiment’ still remains an important tool for future studies (Goto and Numata, 2014). It is known that egg parasitoids of the genus Trichogramma Westw. (Hymenoptera: Trichogrammatidae) are polyvoltine and Trichogramma species of the temperate zone have a facultative prepupal diapause induced by short photoperiods and low temperatures (Boivin, 1994; Laing and Corrigan, 1995; Reznik, 2011). Recently we have demonstrated that short-day conditions during preimaginal development of females of certain Trichogramma species induced a significant increase in the percentage of diapausing prepupae in two or even more generations of progeny and that this short-day effect can be accumulated over several generations (Reznik et al., 2012; Reznik and Ovchinnikov, 2014; Voinovich et al., 2013). The second type of maternal effect (females which underwent diapause are not capable to produce diapausing progeny) was also observed in Trichogramma wasps, but it was not studied in detail (J. Pizzol, pers. comm; Voegelé et al., 1986). In particular, its stability in the sequence of generations was not estimated, whereas the duration of the maternal effect can be essential for identification of its mechanism (‘metabolic’ vs. ‘signal’). Trichogramma wasps are particularly interesting in this regard as the two types of maternal effects on diapause were not often found in one and the same species. In addition, egg parasitoids of the genus Trichogramma are widely used for biological protection of plants and constitute an important beneficial component of natural and agricultural ecosystems (Smith, 1996). The data on the duration of their maternal effect on progeny diapause could be used for development of optimal methods for mass rearing and storage and for the modeling of their seasonal cycles under natural conditions (Boivin, 1994; Parra, 2010). In the present study, we investigated the dynamics of the recovery of the ability to enter diapause in the generation sequence of Trichogramma principium Sug. et Sor. and Trichogramma telengai Sor. (the latter is a parthenogenetic strain of Trichogramma embryophagum Htg. which was described by Sorokina (1987) as a separate species). A strong maternal photoperiodic effect on diapause induction was demonstrated for both species (Reznik et al., 2002; Reznik and Kats, 2004). Experiments with T. principium have also revealed strong grand-maternal and even grand-grandmaternal photoperiodic effects, whereas in T. telengai the grand-maternal effect was very weak (Reznik et al., 2012). Possibly, the reason of this interspecific difference is that T. principium occurs in Southern Europe, Southern Kazakhstan, and Central Asia (i.e., in regions with a warm climate) whereas T. telengai is distributed over northern temperate zone of Eurasia where the wasps develop slowly, have low number of generations per year, enter diapause early, and thus the grandmothers of the diapausing generation develop under long-day conditions (Reznik and Ovchinnikov, 2014; Voinovich et al., 2013). Considering the difference in the average number of generations per year, it can be expected that the diapause-preventing maternal effect in T. principium will be also more stable than in T. telengai. Thus, if the maternal inhibition of diapause in Trichogramma wasps is based on the signal (not metabolic) mechanism and if the parameters of this effect are adapted to the local climate, it can be expected that (1) the prevention of the induction of diapause in the progeny of females that had undergone diapause will persist over two or more generations and (2) in T. principium this
15
effect will be more stable than in T. telengai. The aim of our study was to check these hypotheses. 2. Materials and methods 2.1. Insects, collection sites, and rearing methods The experiments were conducted with a laboratory strain of T. telengai originated from Moscow prov. of Russia and a strain of T. principium originated from Chimkent (Shimkent) prov. of Kazakhstan. The lower thermal threshold for development of the studied strain of T. telengai is ca 10 °C, the sum of effective temperatures (SET) is 190 degree-days; T. principium has a somewhat higher threshold (ca 12 °C) and SET of 150 degree-days (our unpublished data). The climate of Moscow is rather cold: the year average is 6.3 °C and the sum of temperatures above 10 °C is about 900 degree-days that ensures the development of 4–5 generations of T. telengai. The climate of Chimkent is much warmer: the year average is 13.6 °C and the sum of temperatures above 12 °C is about 1800 degree-days that ensures the development of 10–12 generations of T. principium (climate data from: www.weatheronline.co.uk). Both strains were reared during more than 10 years on the eggs of the Angoumois grain moth, Sitotroga cerealella Oliv. (Lepidoptera: Gelechiidae) at the temperature of 20 °C, at the long day (18 h) and at the relative air humidity of ca 75%. The wasps were kept in standard (20 100 mm) test tubes, the grain moth eggs were glued to hard paper cards with non-toxic, water soluble polyvinyl acetate glue; each tube contains 2000–3000 host eggs. Voucher specimens of both strains are kept in the Laboratory of Experimental Entomology, Zoological Institute, St. Petersburg, Russia. 2.2. Experimental methods To start a run of the experiment, two successive generations of each Trichogramma species were reared at the temperature of 20 °C under short day (12 h) conditions, then the next (diapausing) generation was kept for 30 days (from egg to prepupal stage) under the same photoperiod but at the temperature of 12 °C. As expected, this combination of factors ensured induction of diapause in most of individuals. Diapausing prepupae were for 120 days kept in the dark at 5 °C and RH of ca 75% for reactivation after diapause (Hodek, 2002; Laing and Corrigan, 1995) and then the experiment proper was begun. The cards with parasitized host eggs were separated in 4 parts which were placed in different test tubes and then incubated at 20 °C and two photoperiodic regimes: diapause-averting long (18 h) day (Fig. 1A and B) and diapause-inducing short (12 h) day (Fig. 1C), two tubes were used for each photoperiod. Each photoperiod was maintained in two chambers which were periodically replaced within a thermostatic room. The tubes of the same run were always placed in different photoperiodic chambers. After the mass emergence of reactivated adults, 9 small cards and 2 large cards were placed in each tube for 2 h; ca 100 grain moth eggs were glued on each small card and ca 1000 eggs on each large card. Then the cards with just-parasitized hosts were taken from the tubes and all females were immediately removed. The small cards (individuals to be tested for the diapause ability) were distributed among 3 thermal regimes: 12, 13, and 14 °C for T. principium and 13, 14, and 15 °C for T. telengai; these moderately diapause-inducing temperatures were selected based on the results of our earlier studies (Reznik et al., 2002, 2012; Reznik and Ovchinnikov, 2014; Voinovich et al., 2013). Day length (12 h) and air humidity (ca 75%) were
16
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
Fig. 1. The design of the experiment. (A) Long-day sequence of generations (the parental individuals were reared under long-day conditions), (B) long-short-day sequence of generations (the parental individuals were reared under short-day conditions during only one generation directly preceding the testing for the ability to diapause), (C) shortday sequence of generations (the parental individuals were reared under short-day conditions). Numbers of post-diapause generations are indicated at the top of the figure; zero means the diapausing generation (for this generation, photoperiodic and temperature conditions of pupal development are indicated). Photoperiods are indicated as solid (short day, L:D = 12:12) and dashed (long day, L:D = 18:6) rectangles and circles. Temperature (°C) is indicated inside the figures. Arrows indicate the sequence of generations. Rectangles indicate sequential parental generations; circles indicate testing for the ability to diapause (Trichogramma principium was tested at 12–14 °C, Trichogramma telengai was tested at 13–15 °C).
equal in all thermal regimes. The large cards (individuals used as the first post-diapause maternal generation) were placed in new tubes and, in accordance with the design of the experiment (Fig. 1), were incubated at one of the two photoperiodic regimes (12 or 18 h) at 20 °C and RH of ca 75%. The emerged adults of the first generation were also offered small and large cards; the individuals on 9 small cards were again tested for the tendency to diapause and the individuals on large cards were used as the second experimental generation. In this way, 5 generations were obtained. As seen in Fig. 1, the experiment included three sequences of generations: the ‘long-day sequence of generations’ reared under long day conditions (Fig. 1A), the ‘long-short-day sequence of generations’ in which the parental individuals were reared under short-day conditions during only one generation before the test (Fig. 1B), and the ‘short-day sequence of generations’ permanently reared under short day conditions (Fig. 1C). The long-day and short-day sequences included 5 generations tested for the diapause ability (Fig. 1A and C), whereas the long-short-day sequence included 4 tested generations (Fig. 1B). Thus, the experiment included 14 photoperiodic treatments differed in photoperiodic conditions experienced by the current and/or preceding experimental generations, each treatment was represented by two tubes; and in each treatment, the progeny generation was tested for diapause under 3 thermal regimes. In each run of the experiment, each of the 84 resulting combinations (14 photoperiodic treatments 3 thermal regimes 2 tubes) was represented by 3 ‘progeny cards’. Six runs of the experiment were started with an interval of 18–20 days using different generations of laboratory
strains of each Trichogramma species with a total of 1512 progeny cards (more than 150,000 individuals) per species. In 40–60 days (depending on temperature), when the development of the active (non-diapausing) individuals of progeny was completed, all parasitized host eggs were dissected. The number of non-diapausing (adults, much rarer pupae) and diapausing (living prepupae) individuals was recorded separately for each card. The number of emerged adults was estimated by the number of parasitized host eggs with emergence holes. Sporadic individuals that had died at the larval or prepupal stage were not counted. Thus the number of active and diapausing individuals on each progeny card was calculated. 2.3. Statistical treatment As was noted above, the tubes of each run were placed in different chambers (i.e., separated in space) and the runs were separated in time. Thus, a tube with the wasps of the maternal generation was considered as a true replicate and used as the unit for statistical analysis. The proportion of the progeny that entered diapause at each temperature was calculated based on 3 cards per each tube. As a result, the statistical sample size was equal (n = 12) for each combination ‘Trichogramma species photoperiodic treatment thermal regime’. For the primary statistical analysis, the proportions of diapausing individuals were arcsine-square root transformed and treated with ANOVA, whereas medians and quartiles of untransformed data are given in text and figures. In addition, one-way ANOVA with the Tukey HSD test was applied to
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
subsets of the data. For the further statistical analysis, the standardized deviation from the mean proportion of diapausing progeny was calculated (Scheiner and Gurevitch, 2001). For this calculation we used the formula D = 100 (X M)/M, where D is the standardized deviation (%), X is the arcsine-square root transformed proportion of diapausing progeny calculated for a given tube, and M is the mean of arcsine-square root transformed proportions of diapausing progeny calculated for all tubes of a given combination ‘temperature run of the experiment’ (see the Results for the substantiation for this standardization). All the calculations were made with SYSTAT 10.2.
3. Results 3.1. T. principium Three-way ANOVA of the transformed data showed that the proportion of diapausing individuals was highly significantly dependent on photoperiodic treatment (the number of generations after diapause and photoperiodic conditions of their development) and on temperature during development of the progeny (F = 672 and F = 229, correspondingly, both P < 0.001). Differences between runs of the experiment were also significant (F = 151, P < 0.001). The patterns of the changes in the proportion of diapausing progeny tested under different temperatures were very similar (Fig. 2). In addition, interactions between the factors ‘photoperiodic treatment’, ‘temperature’, and ‘run of the experiment’ were much weaker than the separate effects of these factors although also statistically significant (F < 25, P < 0.001). Thus, in order to better reveal the difference among treatments, for the further statistical analysis we used the standardized deviation from the mean proportion of diapausing progeny calculated for a given combination of factors ‘temperature’ and ‘run of the experiment’ (Fig. 3A).
17
First, we note that the proportion of diapausing progeny of females that had undergone diapause was very close to zero (in total, 60 of 17,982 individuals) and was not significantly dependent on photoperiodic conditions of post-diapause development of maternal generation. Moreover, the incidence of diapause in the progeny was very low (less than 1% at any temperature) and only slightly (not significantly) increased when 5 generations were reared under long day conditions (Figs. 2A and 3A). Rearing of one generation under short-day (diapause-inducing) conditions resulted in a slight increase in the incidence of diapause: the difference between the first and second post-diapause generations of the short-day sequence was statistically significant (P = 0.011) only for the standardized data (Figs. 2C and 3A). Then the proportion of diapausing progeny increased gradually with the number of generations after diapause even in the long-short-day sequence, when all generations (excluding the maternal one) were reared under long-day conditions (Figs. 2B and 3A). Finally, in the short-day sequence, when the wasps were constantly reared under short-day conditions, the proportion of diapausing progeny of T. principium significantly increased at least during 5 generations (Figs. 2C and 3A). 3.2. T. telengai Three-way ANOVA of the transformed data showed that the proportion of diapausing individuals of T. telengai also was strongly dependent on photoperiodic conditions experienced by the maternal and grand-maternal generations and on temperature during development of the progeny (F = 1782 and F = 2117, correspondingly, both P < 0.001), whereas differences between runs of the experiment were relatively weak, although statistically significant (F = 10, P < 0.001), as well as all interactions (F < 35, P < 0.001). Thus, the standardized deviation from the mean proportion of diapausing progeny was calculated for this species too.
Fig. 2. The proportion of diapausing progeny of Trichogramma principium in relation to the number of post diapause generations and temperature of development of the progeny tested for the ability to diapause. (A) Long-day sequence of generations, (B) long-short day sequence of generations, (C) short-day sequence of generations. Medians and quartiles of untransformed data (%) are shown; values indicated by different letters are significantly different (P < 0.05 by the Tukey HSD test of arcsine-square root transformed data). Numbers of post-diapause generations correspond to those in the text and Fig. 1; temperature of development of the progeny is shown in the figures.
18
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
Fig. 3. The proportion of diapausing progeny of Trichogramma principium (A) and T. telengai (B) in relation to the number of post-diapause generations and photoperiodic conditions of their development. The standardized deviation from the mean proportion of diapausing progeny is shown (medians and quartiles, %, see Section 2.3 for the formula of calculation). Photoperiodic treatments: 12 h – the sequence of generations reared under short day conditions, 18 ? 12 h – the sequence of generations reared under long-short day conditions (the parental individuals were reared under short-day conditions during only one generation directly preceding the testing for the ability to diapause), 18 h – the sequence of generations reared under long day conditions. Values indicated by different letters are significantly different (P < 0.05 by the Tukey HSD test of arcsine-square root transformed data).
The proportion of diapausing progeny of T. telengai females that had undergone diapause (Fig. 4A and C) was somewhat higher than that in T. principium but still less than 1% (in total, 186 of 20,042 individuals). However, the further dynamics of the proportion of diapausing progeny in sequential generations of the two species was markedly different. First, the inclination to diapause sharply increased over two post-diapause generations. This effect was very strong and statistically significant when the wasps were reared under constant long day conditions (Fig. 4A) and when one or two generations developed under short day (Fig. 4B and C). Second, starting from the third post-diapause generation, the proportion of diapausing individuals was stable: the difference between the 3rd, 4th, and 5th generations was not significant at all the used photoperiodic and thermal regimes (Fig. 4) and the treatment of the pooled standardized data gave the same result (Fig. 3B). 4. Discussion First, we conclude that at the end of the experiment, in 5 generations after diapause, the proportion of diapausing progeny of T. principium and T. telengai was close to that recorded under corresponding photo-thermal regimes in earlier studies (Reznik and Kats, 2004; Reznik and Ovchinnikov, 2014; Reznik et al., 2002,
2012; Sorokina, 1987; Voinovich et al., 2013). This suggests that long-term rearing under laboratory conditions does not significantly change the parameters of the photoperiodic responses of the studied strains. Significant differences among non-synchronous runs of the experiment are also typical of Trichogramma species. Fluctuations in the tendency to diapause, in the pattern of the photo-thermal responses, and in some other biological parameters have been repeatedly observed in successive generations of Trichogramma strains reared under constant conditions and in some cases the range of these fluctuations was even wider than the effect of experimental factors (Reznik, 2011; Schmuck et al., 1996; Vaghina et al., 2014; Zaslavski and Umarova, 1990). The diapause-averting maternal effect has been shown for Trichogramma wasps (Voegelé et al., 1986) although we first demonstrated that it can persist over several generations. It is known that in several insect species the incidence of diapause among the progeny of females that had undergone diapause was relatively low or even zero despite the strong diapause-inducing conditions (Campbell and Tregidga, 2006; Dixon, 1971, 1972; Henrich and Denlinger, 1982; Jackson, 1963; Lees, 1960, 1966; Lushai et al., 1996; Saunders et al., 2002; Saunders, 2010; Simmonds, 1947; Wilson, 1938). In some of the cited studies the stability of this effect was estimated. The experiments with Sarcophaga bullata Parker (in this flesh fly, the diapause-averting effect is induced by the exposure of maternal larvae to short photoperiods) demonstrated that when females had undergone diapause and then developed under the long day during one generation, more than half of their progeny was capable of entering diapause, which was close to the results for the progeny of females constantly reared under long day conditions (Henrich and Denlinger, 1982; Rockey et al., 1989). In other words, in S. bullata, the inability to produce diapausing progeny was stable during only one generation. Similar results were obtained for Lucilia hirsutula Gr. and Calliphora uralensis Vill. (Zinovjeva, 1978). In Megoura viciae Buckton, on the contrary, virginoparous females emerged from diapausing eggs were incapable of producing oviparous females and males over several generations (ca 50 days). Thereafter, the ability to produce sexuales under short day length was rapidly (during 10–20 days) restored. The duration of the period of insensitivity to diapause-inducing factors markedly varied among clones and was dependent on temperature but not on the number of generations passed suggesting the existence of a special ‘interval timer’ (Lees, 1960). The similar results were obtained in experiments with Aphis chloris Koch. Aphis rubicola Oestlund, Drepanosiphum platanoides (Schrank), Eucallipterus tiliae L., Myzus persicae (Sulzer), and Phorodon humuli (Schrank) although in these aphids, the ability to produce sexuales gradually increased over several generations (Brodel and Schaefers, 1979; Campbell and Tregidga, 2006; Dixon, 1971, 1972; Margaritopoulos et al., 2002; Wilson, 1938). It is interesting that we have found both gradual (in T. principium) and relatively rapid (in T. telengai) waning of the maternal restraining effect. In S. bullata, the maternal suppression of diapause was practically absolute: none of more than 3600 individuals entered diapause whereas in females that developed without diapause the proportion of diapausing progeny was more than 50%. In similar experiments with other flies (C. uralensis, L. hirsutula, and Sarcophaga crassipalpis), however, the percentage of diapausing progeny only decreased to various extents (Henrich and Denlinger, 1982; Zinovjeva, 1978). In insect parasitoids, only some decrease in the proportion of diapausing progeny was observed (Jackson, 1963; Simmonds, 1947). In some aphid species the effect was absolute or almost absolute, at least in the first post-diapause generation (Campbell and Tregidga, 2006; Dixon, 1971, 1972; Lees, 1960, 1966; Lushai et al., 1996; Margaritopoulos et al., 2002;
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
19
Fig. 4. The proportion of diapausing progeny of Trichogramma telengai in relation to the number of post diapause generations and temperature of development of the progeny tested for the ability to diapause. (A) Long-day sequence of generations, (B) long-short day sequence of generations, (C) short-day sequence of generations. Explanations as in Fig. 2.
Wilson, 1938). In Trichogramma species, at least under the circumstances of the present study, the diapause-averting maternal effect was also practically absolute. To summarize, we conclude that our first hypothesis was supported by the data: the prevention of the induction of diapause in the progeny of females that had undergone diapause persisted in Trichogramma species over two (in T. telengai) or even five (in T. principium) generations suggesting that the observed effect is based on the signal (not metabolic) mechanism. Until present, multigenerational inability to produce sexual morphs that lay diapause eggs have been thoroughly investigated in aphids (Brodel and Schaefers, 1979; Campbell and Tregidga, 2006; Denlinger, 1998, 2002; Dixon, 1971, 1972; Lees, 1960, 1966; Lushai et al., 1996; Margaritopoulos et al., 2002; Ogawa and Miura, 2014; Saunders et al., 2002). We first described this phenomenon in Hymenoptera, although it is not yet clear is it similar to that in Aphidae, in particular, whether it is based on ‘an interval timer’ or ‘a generation counter’ and is it temperature-compensated or not. The second hypothesis also was not refuted. Considering the adaptive meaning of the maternal inhibition of diapause, we speculated that the stability of this effect, as well as that of the maternal induction of diapause (Reznik and Ovchinnikov, 2014; Voinovich et al., 2013), should increase with the number of generations per year. Indeed, the observed difference between T. principium and T. telengai follows this rule. Although this single fact is not sufficient for a generalization, it should be noted that the most stable effect was observed in aphids (Brodel and Schaefers, 1979; Campbell and Tregidga, 2006; Dixon, 1971, 1972; Lees, 1960, 1966; Margaritopoulos et al., 2002; Saunders et al., 2002; Wilson, 1938) and in Trichogramma wasps (the present study) which under natural conditions are able to produce tens of generations per season. Moreover, both in Trichogramma and in aphids the absolute effect was found: the first post-diapause generation (or even generations) was incapable to enter diapause. In most of other insects studied only a quantitative effect was observed: some proportion of diapausing individuals was found even in the progeny of females that had undergone diapause which can be considered as a
risk-spreading strategy (Henrich and Denlinger, 1982; Jackson, 1963; Simmonds, 1947; Zinovjeva, 1978). Acknowledgements We thank T.Ya. Umarova for the assistance in the experiments. We are deeply grateful to two anonymous reviewers for valuable critical comments and suggestions. References Boivin, G., 1994. Overwintering strategies of egg parasitoids. In: Wajnberg, E., Hassan, S.A. (Eds.), Biological Control with Egg Parasitoids. CAB International, Wallingford, UK, pp. 219–244. Bossdorf, O., Richards, C.L., Pigliucci, M., 2008. Epigenetics for ecologists. Ecol. Lett. 11, 106–115. Brodel, C.F., Schaefers, G.A., 1979. An ‘interval timer’ for the production of oviparae in Aphis rubicola (Homoptera: Aphididae). Entomol. Exp. Appl. 25, 1–8. Campbell, C.A., Tregidga, E.L., 2006. A transgenerational interval timer inhibits unseasonal sexual morph production in damson-hop aphid, Phorodon humuli. Physiol. Entomol. 31, 394–397. Denlinger, D.L., 1998. Maternal control of fly diapause. In: Mousseau, T.A., Fox, C.W. (Eds.), Maternal Effects as Adaptations. Oxford University Press, New York, pp. 275–287. Denlinger, D.L., 2002. Regulation of diapause. Annu. Rev. Entomol. 47, 93–122. Denlinger, D.L., Yocum, G.D., Rinehart, J.P., 2012. Hormonal control of diapause. In: Gilbert, L.I. (Ed.), Insect Endocrinology. Elsevier, Amsterdam, pp. 430–463. Dixon, A.F.G., 1971. The ‘interval timer’ and photoperiod in the determination of parthenogenetic and sexual morphs in the aphid, Drepanosiphum platanoides. J. Insect Physiol. 17, 251–260. Dixon, A.F.G., 1972. The ‘interval timer’, photoperiod and temperature in the seasonal development of parthenogenetic and sexual morphs in the lime aphid, Eucallipterus tiliae L. Oecologia 9, 301–310. Goto, S., Numata, H., 2014. Insect photoperiodism. In: Hoffmann, K.H. (Ed.), Insect Molecular Biology and Ecology. CRC Press, Taylor & Francis Group, Boca Raton, FL, USA, pp. 217–244. Henrich, V.C., Denlinger, D.L., 1982. A maternal effect that eliminates pupal diapause in progeny of the flesh fly, Sarcophaga bullata. J. Insect Physiol. 28, 881–884. Hercus, M.J., Hoffmann, A.A., 2000. Maternal and grandmaternal age influence offspring fitness in Drosophila. Proc. R. Soc. Lond. B 267, 2105–2110. Ho, D.H., Burggren, W.W., 2010. Epigenetics and transgenerational transfer: a physiological perspective. J. Exp. Biol. 213, 3–16. Hodek, I., 2002. Controversial aspects of diapause development. Eur. J. Entomol. 99, 163–174.
20
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
Jablonka, E., Raz, G., 2009. Transgenerational epigenetic inheritance: prevalence, mechanisms, and implications for the study of heredity and evolution. Q. Rev. Biol. 84, 131–176. Jackson, D.J., 1963. Diapause in Caraphractus cinctus Walker (Hymenoptera: Mymaridae), a parasitoid of the eggs of Dytiscidae (Coleoptera). Parasitology 53, 225–251. Kyneb, A., Toft, S., 2006. Effects of maternal diet quality on offspring performance in the rove beetle Tachyporus hypnorum. Ecol. Entomol. 31, 322–330. Laing, J.E., Corrigan, J.E., 1995. Diapause induction and post-diapause emergence in Trichogramma minutum Riley (Hymenoptera: Trichogrammatidae): the role of host species, temperature, and photoperiod. Can. Entomol. 127, 103–110. Lees, A.D., 1960. The role of photoperiod and temperature in the determination of parthogenetic and sexual forms in the aphid Megoura viciae Buckton. II. The operation of the ‘interval timer’ in young clones. J. Insect Physiol. 4, 154–175. Lees, A.D., 1966. The control of polymorphism in aphids. Adv. Insect Physiol. 3, 207– 277. Lushai, G., Hardie, J., Harrington, R., 1996. Inhibition of sexual morph production in the bird cherry aphid, Rhopalosiphum padi. Entomol. Exp. Appl. 81, 117–119. Margaritopoulos, J.T., Tsitsipis, J.A., Prophetou-Athanasiadou, D.A., 2002. An interval timer controls the production of sexual morphs in Myzus persicae (Homoptera: Aphididae). Physiol. Entomol. 27, 251–255. Mousseau, T.A., Fox, C.W., 1998. The adaptive significance of maternal effects. Trends Ecol. Evol. 13, 403–407. Ogawa, K., Miura, T., 2014. Aphid polyphenisms: trans-generational developmental regulation through viviparity. Front. Physiol. 5, 1. http://dx.doi.org/10.3389/ fphys.2014.00001. Parra, J.R.P., 2010. Mass rearing of egg parasitoids for biological control programs. In: Consoli, F.L., Parra, J.R.P., Zucchi, R.A. (Eds.), Egg Parasitoids in Agroecosystems with Emphasis on Trichogramma. Springer, The Netherlands, pp. 267–292. Reznik, S.Ya., 2011. Ecological and evolutionary aspects of photothermal regulation of diapause in trichogrammatidae. J. Evol. Biochem. Physiol. 47, 512–523. Reznik, S.Ya., Kats, T.S., 2004. Exogenous and endogenous factors inducing diapause in Trichogramma principium Sug. et Sor. (Hymenoptera, Trichogrammatidae). Entomol. Rev. 84, 963–970. Reznik, S.Ya., Ovchinnikov, A.N., 2014. Cumulative induction of diapause in successive generations of Trichogramma species (Hymenoptera, Trichogrammatidae). Entomol. Rev. 94, 149–156. Reznik, S.Ya., Kats, T.S., Umarova, T.Ya., Voinovich, N.D., 2002. Maternal age and endogenous variation in maternal influence on photoperiodic response in the progeny diapause in Trichogramma embryophagum (Hymenoptera: Trichogrammatidae). Eur. J. Entomol. 99, 175–180. Reznik, S.Ya., Vaghina, N.P., Voinovich, N.D., 2012. Multigenerational maternal effect on diapause induction in Trichogramma species (Hymenoptera: Trichogrammatidae). Biocontrol Sci. Tech. 22, 429–445.
Rockey, S.J., Miller, B.B., Denlinger, D.L., 1989. A diapause maternal effect in the flesh fly, Sarcophaga bullata: transfer of information from mother to progeny. J. Insect Physiol. 35, 553–558. Saunders, D.S., 2010. Photoperiodism in insects: migration and diapause responses. In: Nelson, R.J., Denlinger, D.L., Somers, D.E. (Eds.), Photoperiodism: The Biological Calendar. Oxford University Press, New York, pp. 218–257. Saunders, D.S., Steel, C.G.H., Vafopoulou, X., Lewis, R.D., 2002. Insect Clocks. Elsevier, Amsterdam. Scheiner, S., Gurevitch, J., 2001. Design and Analysis of Ecological Experiments. Oxford University Press, USA. Schmuck, R., Mager, H., Künast, Ch., Bock, K.-D., Strock-Weyhermüller, S., 1996. Variability in the reproductive performance of beneficial insects in standard laboratory toxicity assays – Implications for hazard classification of pesticides. Ann. Appl. Biol. 128, 437–451. Simmonds, F.J., 1947. Some factors influencing diapause. Can. Entomol. 79, 226– 232. Smith, S.M., 1996. Biological control with Trichogramma: advances, successes, and potential of their use. Annu. Rev. Entomol. 41, 375–406. Sorokina, A.P., 1987. Biological and morphological substantiation of the specific status of Trichogramma telengai sp. n. (Hymenoptera, Trichogrammatidae). Entomol. Obozr. 66, 32–46 (in Russian, English summary). Uller, T., 2008. Developmental plasticity and the evolution of parental effects. Trends Ecol. Evol. 23, 432–438. Vaghina, N.P., Voinovich, N.D., Reznik, S.Ya., 2014. Maternal thermal and photoperiodic effects on the progeny diapause in Trichogramma telengai Sorokina (Hymenoptera: Trichogrammatidae). Entomol. Sci. 17, 198–206. Voegelé, J., Pizzol, J., Raynaud, B., Hawlitzky, N., 1986. La diapause chez les Trichogrammes et ses avantages pour la production de masse et la lutte biologique. Mededelingen Faculteit Landbouwwetenschappen Rijksuniversiteit Gent 51, 1033–1039. Voinovich, N.D., Vaghina, N.P., Reznik, S.Ya., 2013. Comparative analysis of maternal and grand-maternal photoperiodic responses of Trichogramma species (Hymenoptera: Trichogrammatidae). Eur. J. Entomol. 110, 451–460. Wilson, F., 1938. Some experiments on the influence of environment upon the forms of Aphis chloris Koch. (Aphididae). Trans. R. Entomol. Soc. London 87, 165–180. Yamashita, O., 1996. Diapause hormone of the silkworm, Bombyx mori: structure, gene expression and function. J. Insect Physiol. 42, 669–679. Zaslavski, V.A., Umarova, T.Ya., 1990. Environmental and endogenous control of diapause in Trichogramma species. Entomophaga 35, 23–29. Zinovjeva, K.B., 1978. Maternal influence on the induction of larval diapause in Lucilia hirsutula Gr., L. illustris Meig, and Calliphora uralensis Vill. (Diptera, Calliphoridae). Tr. Zool. Inst. 69, 80–94 (in Russian).
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Multigenerational maternal inhibition of prepupal diapause in two Trichogramma species (Hymenoptera: Trichogrammatidae) Sergey Ya. Reznik ⇑, Konstantin G. Samartsev Laboratory of Experimental Entomology, Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia
a r t i c l e
i n f o
Article history: Received 30 March 2015 Received in revised form 1 June 2015 Accepted 23 June 2015 Available online 24 June 2015 Keywords: Diapause Maternal effect Interval timer Photoperiod Trichogramma
a b s t r a c t It is known that in some insect species the incidence of diapause among the progeny of females that had undergone diapause is relatively low or zero even under strong diapause-inducing conditions. Moreover, the maternal inhibition, preventing the induction of a maladaptive diapause in spring, can persist over several generations. This multigenerational effect based on hypothetical ‘interval timer’ was thoroughly studied in Aphididae. We first described a similar phenomenon in Hymenoptera: laboratory experiments demonstrated that the proportion of diapausing progeny of Trichogramma females that had undergone diapause was practically zero independently of photoperiodic and temperature conditions used (day lengths of 12 and 18 h and temperatures of 12–15 °C). Then the ability to enter diapause recovered gradually and returned to the normal level over two (in Trichogramma telengai) or even five (in Trichogramma principium) generations. We conclude that the observed effect may be based on an interval timer similar to that in aphids. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Various maternal effects on diapause induction have been found in many insect species. Based on external manifestations, these effects can be separated into two categories: (1) maternal exposure to diapause-inducing environmental stimuli results in producing diapausing progeny and (2) females that had undergone diapause are incapable of producing diapausing progeny even under strong diapause-inducing conditions. Maternal effects of the first type were often detected when the progeny has no time to react to diapause-inducing stimuli (e.g., in insect species with an embryonic diapause) or cannot perceive environmental cues (e.g., in Diptera and Hymenoptera with larvae living in soil and other cryptic habitats). The reactions of the second type are most probably aimed at preventing the induction of an untimely maladaptive diapause in the progeny of females that had undergone diapause, e.g., when the first spring generation of a polyvoltine species is subjected to late frosts (Denlinger, 1998, 2002; Lees, 1966; Mousseau and Fox, 1998; Ogawa and Miura, 2014; Saunders et al., 2002; Saunders, 2010). In a broad sense, all the above maternal effects can be considered as transgenerational developmental plasticity, but their ⇑ Corresponding author at: Laboratory of Experimental Entomology, Zoological Institute, Russian Academy of Sciences, Universitetskaya nab., 1, St. Petersburg 199034, Russia. E-mail address: [email protected] (S.Ya. Reznik). http://dx.doi.org/10.1016/j.jinsphys.2015.06.012 0022-1910/Ó 2015 Elsevier Ltd. All rights reserved.
physiological mechanisms (in the rare cases when they were revealed) were shown to be very different. In the simplest cases, maternal effects on diapause are based on a direct transfer of nutrients from mother to progeny. For example, in Bombyx mori L. the diapause hormone of the female influences carbohydrate and polyol content of the egg and thus determines diapause of the embryo (Denlinger, 2002; Yamashita, 1996). Such ‘metabolic effects’ resulting from a transfer of nutrients directly influencing the development of progeny were mostly detected in the first generation. In some cases, such effects were found in eggs but never in larvae of the second generation because the nutrient was too much diluted (Hercus and Hoffmann, 2000; Kyneb and Toft, 2006). The ‘signal effects’ are based either on the transmission of cytoplasmic factors (RNA, hormones) or on the epigenetic inheritance in the strict sense of the term (transgenerational transmission of variations in DNA expression). It is well known that environmentally induced epigenetic changes ensuring transmission of phenotypic characters without modification to gene sequences can be often traced over several generations (Bossdorf et al., 2008; Ho and Burggren, 2010; Jablonka and Raz, 2009; Uller, 2008). Of course, metabolic and signal mechanisms are not mutually exclusive, but long-term effects are most probably based on transfer of signals rather than nutrients. In the regulation of insect diapause, these effects are often manifested as ‘interval timers’ which measure time intervals independently of the number of elapsed generations, although ‘generation counters’ reducing the effect in accordance
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
with the number of generations passed can be also conceived (Brodel and Schaefers, 1979; Campbell and Tregidga, 2006; Denlinger, 1998, 2002; Dixon, 1971, 1972; Lees, 1960, 1966; Lushai et al., 1996; Margaritopoulos et al., 2002; Ogawa and Miura, 2014; Saunders et al., 2002). At present, biochemical and molecular mechanisms of diapause induction are intensively investigated (Denlinger, 2002; Denlinger et al., 2012) but ‘classic physiological experiment’ still remains an important tool for future studies (Goto and Numata, 2014). It is known that egg parasitoids of the genus Trichogramma Westw. (Hymenoptera: Trichogrammatidae) are polyvoltine and Trichogramma species of the temperate zone have a facultative prepupal diapause induced by short photoperiods and low temperatures (Boivin, 1994; Laing and Corrigan, 1995; Reznik, 2011). Recently we have demonstrated that short-day conditions during preimaginal development of females of certain Trichogramma species induced a significant increase in the percentage of diapausing prepupae in two or even more generations of progeny and that this short-day effect can be accumulated over several generations (Reznik et al., 2012; Reznik and Ovchinnikov, 2014; Voinovich et al., 2013). The second type of maternal effect (females which underwent diapause are not capable to produce diapausing progeny) was also observed in Trichogramma wasps, but it was not studied in detail (J. Pizzol, pers. comm; Voegelé et al., 1986). In particular, its stability in the sequence of generations was not estimated, whereas the duration of the maternal effect can be essential for identification of its mechanism (‘metabolic’ vs. ‘signal’). Trichogramma wasps are particularly interesting in this regard as the two types of maternal effects on diapause were not often found in one and the same species. In addition, egg parasitoids of the genus Trichogramma are widely used for biological protection of plants and constitute an important beneficial component of natural and agricultural ecosystems (Smith, 1996). The data on the duration of their maternal effect on progeny diapause could be used for development of optimal methods for mass rearing and storage and for the modeling of their seasonal cycles under natural conditions (Boivin, 1994; Parra, 2010). In the present study, we investigated the dynamics of the recovery of the ability to enter diapause in the generation sequence of Trichogramma principium Sug. et Sor. and Trichogramma telengai Sor. (the latter is a parthenogenetic strain of Trichogramma embryophagum Htg. which was described by Sorokina (1987) as a separate species). A strong maternal photoperiodic effect on diapause induction was demonstrated for both species (Reznik et al., 2002; Reznik and Kats, 2004). Experiments with T. principium have also revealed strong grand-maternal and even grand-grandmaternal photoperiodic effects, whereas in T. telengai the grand-maternal effect was very weak (Reznik et al., 2012). Possibly, the reason of this interspecific difference is that T. principium occurs in Southern Europe, Southern Kazakhstan, and Central Asia (i.e., in regions with a warm climate) whereas T. telengai is distributed over northern temperate zone of Eurasia where the wasps develop slowly, have low number of generations per year, enter diapause early, and thus the grandmothers of the diapausing generation develop under long-day conditions (Reznik and Ovchinnikov, 2014; Voinovich et al., 2013). Considering the difference in the average number of generations per year, it can be expected that the diapause-preventing maternal effect in T. principium will be also more stable than in T. telengai. Thus, if the maternal inhibition of diapause in Trichogramma wasps is based on the signal (not metabolic) mechanism and if the parameters of this effect are adapted to the local climate, it can be expected that (1) the prevention of the induction of diapause in the progeny of females that had undergone diapause will persist over two or more generations and (2) in T. principium this
15
effect will be more stable than in T. telengai. The aim of our study was to check these hypotheses. 2. Materials and methods 2.1. Insects, collection sites, and rearing methods The experiments were conducted with a laboratory strain of T. telengai originated from Moscow prov. of Russia and a strain of T. principium originated from Chimkent (Shimkent) prov. of Kazakhstan. The lower thermal threshold for development of the studied strain of T. telengai is ca 10 °C, the sum of effective temperatures (SET) is 190 degree-days; T. principium has a somewhat higher threshold (ca 12 °C) and SET of 150 degree-days (our unpublished data). The climate of Moscow is rather cold: the year average is 6.3 °C and the sum of temperatures above 10 °C is about 900 degree-days that ensures the development of 4–5 generations of T. telengai. The climate of Chimkent is much warmer: the year average is 13.6 °C and the sum of temperatures above 12 °C is about 1800 degree-days that ensures the development of 10–12 generations of T. principium (climate data from: www.weatheronline.co.uk). Both strains were reared during more than 10 years on the eggs of the Angoumois grain moth, Sitotroga cerealella Oliv. (Lepidoptera: Gelechiidae) at the temperature of 20 °C, at the long day (18 h) and at the relative air humidity of ca 75%. The wasps were kept in standard (20 100 mm) test tubes, the grain moth eggs were glued to hard paper cards with non-toxic, water soluble polyvinyl acetate glue; each tube contains 2000–3000 host eggs. Voucher specimens of both strains are kept in the Laboratory of Experimental Entomology, Zoological Institute, St. Petersburg, Russia. 2.2. Experimental methods To start a run of the experiment, two successive generations of each Trichogramma species were reared at the temperature of 20 °C under short day (12 h) conditions, then the next (diapausing) generation was kept for 30 days (from egg to prepupal stage) under the same photoperiod but at the temperature of 12 °C. As expected, this combination of factors ensured induction of diapause in most of individuals. Diapausing prepupae were for 120 days kept in the dark at 5 °C and RH of ca 75% for reactivation after diapause (Hodek, 2002; Laing and Corrigan, 1995) and then the experiment proper was begun. The cards with parasitized host eggs were separated in 4 parts which were placed in different test tubes and then incubated at 20 °C and two photoperiodic regimes: diapause-averting long (18 h) day (Fig. 1A and B) and diapause-inducing short (12 h) day (Fig. 1C), two tubes were used for each photoperiod. Each photoperiod was maintained in two chambers which were periodically replaced within a thermostatic room. The tubes of the same run were always placed in different photoperiodic chambers. After the mass emergence of reactivated adults, 9 small cards and 2 large cards were placed in each tube for 2 h; ca 100 grain moth eggs were glued on each small card and ca 1000 eggs on each large card. Then the cards with just-parasitized hosts were taken from the tubes and all females were immediately removed. The small cards (individuals to be tested for the diapause ability) were distributed among 3 thermal regimes: 12, 13, and 14 °C for T. principium and 13, 14, and 15 °C for T. telengai; these moderately diapause-inducing temperatures were selected based on the results of our earlier studies (Reznik et al., 2002, 2012; Reznik and Ovchinnikov, 2014; Voinovich et al., 2013). Day length (12 h) and air humidity (ca 75%) were
16
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
Fig. 1. The design of the experiment. (A) Long-day sequence of generations (the parental individuals were reared under long-day conditions), (B) long-short-day sequence of generations (the parental individuals were reared under short-day conditions during only one generation directly preceding the testing for the ability to diapause), (C) shortday sequence of generations (the parental individuals were reared under short-day conditions). Numbers of post-diapause generations are indicated at the top of the figure; zero means the diapausing generation (for this generation, photoperiodic and temperature conditions of pupal development are indicated). Photoperiods are indicated as solid (short day, L:D = 12:12) and dashed (long day, L:D = 18:6) rectangles and circles. Temperature (°C) is indicated inside the figures. Arrows indicate the sequence of generations. Rectangles indicate sequential parental generations; circles indicate testing for the ability to diapause (Trichogramma principium was tested at 12–14 °C, Trichogramma telengai was tested at 13–15 °C).
equal in all thermal regimes. The large cards (individuals used as the first post-diapause maternal generation) were placed in new tubes and, in accordance with the design of the experiment (Fig. 1), were incubated at one of the two photoperiodic regimes (12 or 18 h) at 20 °C and RH of ca 75%. The emerged adults of the first generation were also offered small and large cards; the individuals on 9 small cards were again tested for the tendency to diapause and the individuals on large cards were used as the second experimental generation. In this way, 5 generations were obtained. As seen in Fig. 1, the experiment included three sequences of generations: the ‘long-day sequence of generations’ reared under long day conditions (Fig. 1A), the ‘long-short-day sequence of generations’ in which the parental individuals were reared under short-day conditions during only one generation before the test (Fig. 1B), and the ‘short-day sequence of generations’ permanently reared under short day conditions (Fig. 1C). The long-day and short-day sequences included 5 generations tested for the diapause ability (Fig. 1A and C), whereas the long-short-day sequence included 4 tested generations (Fig. 1B). Thus, the experiment included 14 photoperiodic treatments differed in photoperiodic conditions experienced by the current and/or preceding experimental generations, each treatment was represented by two tubes; and in each treatment, the progeny generation was tested for diapause under 3 thermal regimes. In each run of the experiment, each of the 84 resulting combinations (14 photoperiodic treatments 3 thermal regimes 2 tubes) was represented by 3 ‘progeny cards’. Six runs of the experiment were started with an interval of 18–20 days using different generations of laboratory
strains of each Trichogramma species with a total of 1512 progeny cards (more than 150,000 individuals) per species. In 40–60 days (depending on temperature), when the development of the active (non-diapausing) individuals of progeny was completed, all parasitized host eggs were dissected. The number of non-diapausing (adults, much rarer pupae) and diapausing (living prepupae) individuals was recorded separately for each card. The number of emerged adults was estimated by the number of parasitized host eggs with emergence holes. Sporadic individuals that had died at the larval or prepupal stage were not counted. Thus the number of active and diapausing individuals on each progeny card was calculated. 2.3. Statistical treatment As was noted above, the tubes of each run were placed in different chambers (i.e., separated in space) and the runs were separated in time. Thus, a tube with the wasps of the maternal generation was considered as a true replicate and used as the unit for statistical analysis. The proportion of the progeny that entered diapause at each temperature was calculated based on 3 cards per each tube. As a result, the statistical sample size was equal (n = 12) for each combination ‘Trichogramma species photoperiodic treatment thermal regime’. For the primary statistical analysis, the proportions of diapausing individuals were arcsine-square root transformed and treated with ANOVA, whereas medians and quartiles of untransformed data are given in text and figures. In addition, one-way ANOVA with the Tukey HSD test was applied to
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
subsets of the data. For the further statistical analysis, the standardized deviation from the mean proportion of diapausing progeny was calculated (Scheiner and Gurevitch, 2001). For this calculation we used the formula D = 100 (X M)/M, where D is the standardized deviation (%), X is the arcsine-square root transformed proportion of diapausing progeny calculated for a given tube, and M is the mean of arcsine-square root transformed proportions of diapausing progeny calculated for all tubes of a given combination ‘temperature run of the experiment’ (see the Results for the substantiation for this standardization). All the calculations were made with SYSTAT 10.2.
3. Results 3.1. T. principium Three-way ANOVA of the transformed data showed that the proportion of diapausing individuals was highly significantly dependent on photoperiodic treatment (the number of generations after diapause and photoperiodic conditions of their development) and on temperature during development of the progeny (F = 672 and F = 229, correspondingly, both P < 0.001). Differences between runs of the experiment were also significant (F = 151, P < 0.001). The patterns of the changes in the proportion of diapausing progeny tested under different temperatures were very similar (Fig. 2). In addition, interactions between the factors ‘photoperiodic treatment’, ‘temperature’, and ‘run of the experiment’ were much weaker than the separate effects of these factors although also statistically significant (F < 25, P < 0.001). Thus, in order to better reveal the difference among treatments, for the further statistical analysis we used the standardized deviation from the mean proportion of diapausing progeny calculated for a given combination of factors ‘temperature’ and ‘run of the experiment’ (Fig. 3A).
17
First, we note that the proportion of diapausing progeny of females that had undergone diapause was very close to zero (in total, 60 of 17,982 individuals) and was not significantly dependent on photoperiodic conditions of post-diapause development of maternal generation. Moreover, the incidence of diapause in the progeny was very low (less than 1% at any temperature) and only slightly (not significantly) increased when 5 generations were reared under long day conditions (Figs. 2A and 3A). Rearing of one generation under short-day (diapause-inducing) conditions resulted in a slight increase in the incidence of diapause: the difference between the first and second post-diapause generations of the short-day sequence was statistically significant (P = 0.011) only for the standardized data (Figs. 2C and 3A). Then the proportion of diapausing progeny increased gradually with the number of generations after diapause even in the long-short-day sequence, when all generations (excluding the maternal one) were reared under long-day conditions (Figs. 2B and 3A). Finally, in the short-day sequence, when the wasps were constantly reared under short-day conditions, the proportion of diapausing progeny of T. principium significantly increased at least during 5 generations (Figs. 2C and 3A). 3.2. T. telengai Three-way ANOVA of the transformed data showed that the proportion of diapausing individuals of T. telengai also was strongly dependent on photoperiodic conditions experienced by the maternal and grand-maternal generations and on temperature during development of the progeny (F = 1782 and F = 2117, correspondingly, both P < 0.001), whereas differences between runs of the experiment were relatively weak, although statistically significant (F = 10, P < 0.001), as well as all interactions (F < 35, P < 0.001). Thus, the standardized deviation from the mean proportion of diapausing progeny was calculated for this species too.
Fig. 2. The proportion of diapausing progeny of Trichogramma principium in relation to the number of post diapause generations and temperature of development of the progeny tested for the ability to diapause. (A) Long-day sequence of generations, (B) long-short day sequence of generations, (C) short-day sequence of generations. Medians and quartiles of untransformed data (%) are shown; values indicated by different letters are significantly different (P < 0.05 by the Tukey HSD test of arcsine-square root transformed data). Numbers of post-diapause generations correspond to those in the text and Fig. 1; temperature of development of the progeny is shown in the figures.
18
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
Fig. 3. The proportion of diapausing progeny of Trichogramma principium (A) and T. telengai (B) in relation to the number of post-diapause generations and photoperiodic conditions of their development. The standardized deviation from the mean proportion of diapausing progeny is shown (medians and quartiles, %, see Section 2.3 for the formula of calculation). Photoperiodic treatments: 12 h – the sequence of generations reared under short day conditions, 18 ? 12 h – the sequence of generations reared under long-short day conditions (the parental individuals were reared under short-day conditions during only one generation directly preceding the testing for the ability to diapause), 18 h – the sequence of generations reared under long day conditions. Values indicated by different letters are significantly different (P < 0.05 by the Tukey HSD test of arcsine-square root transformed data).
The proportion of diapausing progeny of T. telengai females that had undergone diapause (Fig. 4A and C) was somewhat higher than that in T. principium but still less than 1% (in total, 186 of 20,042 individuals). However, the further dynamics of the proportion of diapausing progeny in sequential generations of the two species was markedly different. First, the inclination to diapause sharply increased over two post-diapause generations. This effect was very strong and statistically significant when the wasps were reared under constant long day conditions (Fig. 4A) and when one or two generations developed under short day (Fig. 4B and C). Second, starting from the third post-diapause generation, the proportion of diapausing individuals was stable: the difference between the 3rd, 4th, and 5th generations was not significant at all the used photoperiodic and thermal regimes (Fig. 4) and the treatment of the pooled standardized data gave the same result (Fig. 3B). 4. Discussion First, we conclude that at the end of the experiment, in 5 generations after diapause, the proportion of diapausing progeny of T. principium and T. telengai was close to that recorded under corresponding photo-thermal regimes in earlier studies (Reznik and Kats, 2004; Reznik and Ovchinnikov, 2014; Reznik et al., 2002,
2012; Sorokina, 1987; Voinovich et al., 2013). This suggests that long-term rearing under laboratory conditions does not significantly change the parameters of the photoperiodic responses of the studied strains. Significant differences among non-synchronous runs of the experiment are also typical of Trichogramma species. Fluctuations in the tendency to diapause, in the pattern of the photo-thermal responses, and in some other biological parameters have been repeatedly observed in successive generations of Trichogramma strains reared under constant conditions and in some cases the range of these fluctuations was even wider than the effect of experimental factors (Reznik, 2011; Schmuck et al., 1996; Vaghina et al., 2014; Zaslavski and Umarova, 1990). The diapause-averting maternal effect has been shown for Trichogramma wasps (Voegelé et al., 1986) although we first demonstrated that it can persist over several generations. It is known that in several insect species the incidence of diapause among the progeny of females that had undergone diapause was relatively low or even zero despite the strong diapause-inducing conditions (Campbell and Tregidga, 2006; Dixon, 1971, 1972; Henrich and Denlinger, 1982; Jackson, 1963; Lees, 1960, 1966; Lushai et al., 1996; Saunders et al., 2002; Saunders, 2010; Simmonds, 1947; Wilson, 1938). In some of the cited studies the stability of this effect was estimated. The experiments with Sarcophaga bullata Parker (in this flesh fly, the diapause-averting effect is induced by the exposure of maternal larvae to short photoperiods) demonstrated that when females had undergone diapause and then developed under the long day during one generation, more than half of their progeny was capable of entering diapause, which was close to the results for the progeny of females constantly reared under long day conditions (Henrich and Denlinger, 1982; Rockey et al., 1989). In other words, in S. bullata, the inability to produce diapausing progeny was stable during only one generation. Similar results were obtained for Lucilia hirsutula Gr. and Calliphora uralensis Vill. (Zinovjeva, 1978). In Megoura viciae Buckton, on the contrary, virginoparous females emerged from diapausing eggs were incapable of producing oviparous females and males over several generations (ca 50 days). Thereafter, the ability to produce sexuales under short day length was rapidly (during 10–20 days) restored. The duration of the period of insensitivity to diapause-inducing factors markedly varied among clones and was dependent on temperature but not on the number of generations passed suggesting the existence of a special ‘interval timer’ (Lees, 1960). The similar results were obtained in experiments with Aphis chloris Koch. Aphis rubicola Oestlund, Drepanosiphum platanoides (Schrank), Eucallipterus tiliae L., Myzus persicae (Sulzer), and Phorodon humuli (Schrank) although in these aphids, the ability to produce sexuales gradually increased over several generations (Brodel and Schaefers, 1979; Campbell and Tregidga, 2006; Dixon, 1971, 1972; Margaritopoulos et al., 2002; Wilson, 1938). It is interesting that we have found both gradual (in T. principium) and relatively rapid (in T. telengai) waning of the maternal restraining effect. In S. bullata, the maternal suppression of diapause was practically absolute: none of more than 3600 individuals entered diapause whereas in females that developed without diapause the proportion of diapausing progeny was more than 50%. In similar experiments with other flies (C. uralensis, L. hirsutula, and Sarcophaga crassipalpis), however, the percentage of diapausing progeny only decreased to various extents (Henrich and Denlinger, 1982; Zinovjeva, 1978). In insect parasitoids, only some decrease in the proportion of diapausing progeny was observed (Jackson, 1963; Simmonds, 1947). In some aphid species the effect was absolute or almost absolute, at least in the first post-diapause generation (Campbell and Tregidga, 2006; Dixon, 1971, 1972; Lees, 1960, 1966; Lushai et al., 1996; Margaritopoulos et al., 2002;
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
19
Fig. 4. The proportion of diapausing progeny of Trichogramma telengai in relation to the number of post diapause generations and temperature of development of the progeny tested for the ability to diapause. (A) Long-day sequence of generations, (B) long-short day sequence of generations, (C) short-day sequence of generations. Explanations as in Fig. 2.
Wilson, 1938). In Trichogramma species, at least under the circumstances of the present study, the diapause-averting maternal effect was also practically absolute. To summarize, we conclude that our first hypothesis was supported by the data: the prevention of the induction of diapause in the progeny of females that had undergone diapause persisted in Trichogramma species over two (in T. telengai) or even five (in T. principium) generations suggesting that the observed effect is based on the signal (not metabolic) mechanism. Until present, multigenerational inability to produce sexual morphs that lay diapause eggs have been thoroughly investigated in aphids (Brodel and Schaefers, 1979; Campbell and Tregidga, 2006; Denlinger, 1998, 2002; Dixon, 1971, 1972; Lees, 1960, 1966; Lushai et al., 1996; Margaritopoulos et al., 2002; Ogawa and Miura, 2014; Saunders et al., 2002). We first described this phenomenon in Hymenoptera, although it is not yet clear is it similar to that in Aphidae, in particular, whether it is based on ‘an interval timer’ or ‘a generation counter’ and is it temperature-compensated or not. The second hypothesis also was not refuted. Considering the adaptive meaning of the maternal inhibition of diapause, we speculated that the stability of this effect, as well as that of the maternal induction of diapause (Reznik and Ovchinnikov, 2014; Voinovich et al., 2013), should increase with the number of generations per year. Indeed, the observed difference between T. principium and T. telengai follows this rule. Although this single fact is not sufficient for a generalization, it should be noted that the most stable effect was observed in aphids (Brodel and Schaefers, 1979; Campbell and Tregidga, 2006; Dixon, 1971, 1972; Lees, 1960, 1966; Margaritopoulos et al., 2002; Saunders et al., 2002; Wilson, 1938) and in Trichogramma wasps (the present study) which under natural conditions are able to produce tens of generations per season. Moreover, both in Trichogramma and in aphids the absolute effect was found: the first post-diapause generation (or even generations) was incapable to enter diapause. In most of other insects studied only a quantitative effect was observed: some proportion of diapausing individuals was found even in the progeny of females that had undergone diapause which can be considered as a
risk-spreading strategy (Henrich and Denlinger, 1982; Jackson, 1963; Simmonds, 1947; Zinovjeva, 1978). Acknowledgements We thank T.Ya. Umarova for the assistance in the experiments. We are deeply grateful to two anonymous reviewers for valuable critical comments and suggestions. References Boivin, G., 1994. Overwintering strategies of egg parasitoids. In: Wajnberg, E., Hassan, S.A. (Eds.), Biological Control with Egg Parasitoids. CAB International, Wallingford, UK, pp. 219–244. Bossdorf, O., Richards, C.L., Pigliucci, M., 2008. Epigenetics for ecologists. Ecol. Lett. 11, 106–115. Brodel, C.F., Schaefers, G.A., 1979. An ‘interval timer’ for the production of oviparae in Aphis rubicola (Homoptera: Aphididae). Entomol. Exp. Appl. 25, 1–8. Campbell, C.A., Tregidga, E.L., 2006. A transgenerational interval timer inhibits unseasonal sexual morph production in damson-hop aphid, Phorodon humuli. Physiol. Entomol. 31, 394–397. Denlinger, D.L., 1998. Maternal control of fly diapause. In: Mousseau, T.A., Fox, C.W. (Eds.), Maternal Effects as Adaptations. Oxford University Press, New York, pp. 275–287. Denlinger, D.L., 2002. Regulation of diapause. Annu. Rev. Entomol. 47, 93–122. Denlinger, D.L., Yocum, G.D., Rinehart, J.P., 2012. Hormonal control of diapause. In: Gilbert, L.I. (Ed.), Insect Endocrinology. Elsevier, Amsterdam, pp. 430–463. Dixon, A.F.G., 1971. The ‘interval timer’ and photoperiod in the determination of parthenogenetic and sexual morphs in the aphid, Drepanosiphum platanoides. J. Insect Physiol. 17, 251–260. Dixon, A.F.G., 1972. The ‘interval timer’, photoperiod and temperature in the seasonal development of parthenogenetic and sexual morphs in the lime aphid, Eucallipterus tiliae L. Oecologia 9, 301–310. Goto, S., Numata, H., 2014. Insect photoperiodism. In: Hoffmann, K.H. (Ed.), Insect Molecular Biology and Ecology. CRC Press, Taylor & Francis Group, Boca Raton, FL, USA, pp. 217–244. Henrich, V.C., Denlinger, D.L., 1982. A maternal effect that eliminates pupal diapause in progeny of the flesh fly, Sarcophaga bullata. J. Insect Physiol. 28, 881–884. Hercus, M.J., Hoffmann, A.A., 2000. Maternal and grandmaternal age influence offspring fitness in Drosophila. Proc. R. Soc. Lond. B 267, 2105–2110. Ho, D.H., Burggren, W.W., 2010. Epigenetics and transgenerational transfer: a physiological perspective. J. Exp. Biol. 213, 3–16. Hodek, I., 2002. Controversial aspects of diapause development. Eur. J. Entomol. 99, 163–174.
20
S.Ya. Reznik, K.G. Samartsev / Journal of Insect Physiology 81 (2015) 14–20
Jablonka, E., Raz, G., 2009. Transgenerational epigenetic inheritance: prevalence, mechanisms, and implications for the study of heredity and evolution. Q. Rev. Biol. 84, 131–176. Jackson, D.J., 1963. Diapause in Caraphractus cinctus Walker (Hymenoptera: Mymaridae), a parasitoid of the eggs of Dytiscidae (Coleoptera). Parasitology 53, 225–251. Kyneb, A., Toft, S., 2006. Effects of maternal diet quality on offspring performance in the rove beetle Tachyporus hypnorum. Ecol. Entomol. 31, 322–330. Laing, J.E., Corrigan, J.E., 1995. Diapause induction and post-diapause emergence in Trichogramma minutum Riley (Hymenoptera: Trichogrammatidae): the role of host species, temperature, and photoperiod. Can. Entomol. 127, 103–110. Lees, A.D., 1960. The role of photoperiod and temperature in the determination of parthogenetic and sexual forms in the aphid Megoura viciae Buckton. II. The operation of the ‘interval timer’ in young clones. J. Insect Physiol. 4, 154–175. Lees, A.D., 1966. The control of polymorphism in aphids. Adv. Insect Physiol. 3, 207– 277. Lushai, G., Hardie, J., Harrington, R., 1996. Inhibition of sexual morph production in the bird cherry aphid, Rhopalosiphum padi. Entomol. Exp. Appl. 81, 117–119. Margaritopoulos, J.T., Tsitsipis, J.A., Prophetou-Athanasiadou, D.A., 2002. An interval timer controls the production of sexual morphs in Myzus persicae (Homoptera: Aphididae). Physiol. Entomol. 27, 251–255. Mousseau, T.A., Fox, C.W., 1998. The adaptive significance of maternal effects. Trends Ecol. Evol. 13, 403–407. Ogawa, K., Miura, T., 2014. Aphid polyphenisms: trans-generational developmental regulation through viviparity. Front. Physiol. 5, 1. http://dx.doi.org/10.3389/ fphys.2014.00001. Parra, J.R.P., 2010. Mass rearing of egg parasitoids for biological control programs. In: Consoli, F.L., Parra, J.R.P., Zucchi, R.A. (Eds.), Egg Parasitoids in Agroecosystems with Emphasis on Trichogramma. Springer, The Netherlands, pp. 267–292. Reznik, S.Ya., 2011. Ecological and evolutionary aspects of photothermal regulation of diapause in trichogrammatidae. J. Evol. Biochem. Physiol. 47, 512–523. Reznik, S.Ya., Kats, T.S., 2004. Exogenous and endogenous factors inducing diapause in Trichogramma principium Sug. et Sor. (Hymenoptera, Trichogrammatidae). Entomol. Rev. 84, 963–970. Reznik, S.Ya., Ovchinnikov, A.N., 2014. Cumulative induction of diapause in successive generations of Trichogramma species (Hymenoptera, Trichogrammatidae). Entomol. Rev. 94, 149–156. Reznik, S.Ya., Kats, T.S., Umarova, T.Ya., Voinovich, N.D., 2002. Maternal age and endogenous variation in maternal influence on photoperiodic response in the progeny diapause in Trichogramma embryophagum (Hymenoptera: Trichogrammatidae). Eur. J. Entomol. 99, 175–180. Reznik, S.Ya., Vaghina, N.P., Voinovich, N.D., 2012. Multigenerational maternal effect on diapause induction in Trichogramma species (Hymenoptera: Trichogrammatidae). Biocontrol Sci. Tech. 22, 429–445.
Rockey, S.J., Miller, B.B., Denlinger, D.L., 1989. A diapause maternal effect in the flesh fly, Sarcophaga bullata: transfer of information from mother to progeny. J. Insect Physiol. 35, 553–558. Saunders, D.S., 2010. Photoperiodism in insects: migration and diapause responses. In: Nelson, R.J., Denlinger, D.L., Somers, D.E. (Eds.), Photoperiodism: The Biological Calendar. Oxford University Press, New York, pp. 218–257. Saunders, D.S., Steel, C.G.H., Vafopoulou, X., Lewis, R.D., 2002. Insect Clocks. Elsevier, Amsterdam. Scheiner, S., Gurevitch, J., 2001. Design and Analysis of Ecological Experiments. Oxford University Press, USA. Schmuck, R., Mager, H., Künast, Ch., Bock, K.-D., Strock-Weyhermüller, S., 1996. Variability in the reproductive performance of beneficial insects in standard laboratory toxicity assays – Implications for hazard classification of pesticides. Ann. Appl. Biol. 128, 437–451. Simmonds, F.J., 1947. Some factors influencing diapause. Can. Entomol. 79, 226– 232. Smith, S.M., 1996. Biological control with Trichogramma: advances, successes, and potential of their use. Annu. Rev. Entomol. 41, 375–406. Sorokina, A.P., 1987. Biological and morphological substantiation of the specific status of Trichogramma telengai sp. n. (Hymenoptera, Trichogrammatidae). Entomol. Obozr. 66, 32–46 (in Russian, English summary). Uller, T., 2008. Developmental plasticity and the evolution of parental effects. Trends Ecol. Evol. 23, 432–438. Vaghina, N.P., Voinovich, N.D., Reznik, S.Ya., 2014. Maternal thermal and photoperiodic effects on the progeny diapause in Trichogramma telengai Sorokina (Hymenoptera: Trichogrammatidae). Entomol. Sci. 17, 198–206. Voegelé, J., Pizzol, J., Raynaud, B., Hawlitzky, N., 1986. La diapause chez les Trichogrammes et ses avantages pour la production de masse et la lutte biologique. Mededelingen Faculteit Landbouwwetenschappen Rijksuniversiteit Gent 51, 1033–1039. Voinovich, N.D., Vaghina, N.P., Reznik, S.Ya., 2013. Comparative analysis of maternal and grand-maternal photoperiodic responses of Trichogramma species (Hymenoptera: Trichogrammatidae). Eur. J. Entomol. 110, 451–460. Wilson, F., 1938. Some experiments on the influence of environment upon the forms of Aphis chloris Koch. (Aphididae). Trans. R. Entomol. Soc. London 87, 165–180. Yamashita, O., 1996. Diapause hormone of the silkworm, Bombyx mori: structure, gene expression and function. J. Insect Physiol. 42, 669–679. Zaslavski, V.A., Umarova, T.Ya., 1990. Environmental and endogenous control of diapause in Trichogramma species. Entomophaga 35, 23–29. Zinovjeva, K.B., 1978. Maternal influence on the induction of larval diapause in Lucilia hirsutula Gr., L. illustris Meig, and Calliphora uralensis Vill. (Diptera, Calliphoridae). Tr. Zool. Inst. 69, 80–94 (in Russian).