Individual variation, which is observed within natural populations or within laboratory-reared and industrial strains, affects, to a varying degree, all the morphological, physiological and behavioral traits of insects. Nonhereditary (environmentally induced) variation is the key mechanism of adaptation to relatively short-term changes and spatial heterogeneity of the environment. Hereditary individual variation is the necessary prerequisite to both natural evolution of insects and artificial selection of new lines and varieties of beneficial species. However, uncontrolled intrastrain variation during laboratory and industrial rearing makes it more difficult to carry out research and standardize methods, which not infrequently results in decreased quality of biocontrol agents. Thus, the diverse forms of individual variation warrant further study as being of interest to both basic and applied entomology.

In particular, most insects studied for facultative diapause induction exhibit a more or less substantial intraspecific variation in their responses to diapause-inducing photoperiods and temperatures. Diapause is among the most important ecophysiological adaptations and synchronizes insect seasonal cycles of activity with the dynamics of environmental factors. Facultative diapause usually commences prior to the onset of adverse conditions and is induced by seasonal cues such as photoperiod (day length), temperature, diet quality, etc. Intraspecific variation, both within (individual) and between populations, which underpins the plasticity of diapause responses, provides the basis for risk-spreading strategies and microevolution of seasonal adaptations in insects (Danilevskii, 1961; Zaslavski, 1984; Tauber et al., 1986; Saulich, 1999; Denlinger, 2002; Saunders et al., 2002; Saulich and Volkovich, 2004; Danks, 2007; Tougeron, 2019; Saunders, 2020; Snell-Rood and Ehlman, 2021).

Species of the genus Trichogramma Westw. (Hymenoptera, Trichogrammatidae) are minute parasitoids of eggs and serve as natural enemies to many lepidopteran pests of agriculture and forestry. These wasps are widely used as biological control agents in plant protection (Smith, 1996; Sorokina, 2011). Facultative prepupal winter diapause in Trichogramma is primarily controlled by temperature, but at near-threshold temperatures, the percentage of diapausing progeny strongly depends on the photoperiodic conditions experienced by the maternal generation (Zaslavski and Umarova, 1981; Mai Phu Qui and Zaslavski, 1983; Zaslavski and Umarova, 1990; Boivin, 1994; Reznik, 2011). Different forms of interpopulational (interstrain) variation in photoperiodically and thermally induced diapause responses were discovered in many species of Trichogramma (Sorokina, 1987; Sorokina and Maslennikova, 1987; Sorokina, 2010). However, to the best of our knowledge, no dedicated analysis of intrapopulational (intrastrain) variation has been carried out before our previous work (Reznik and Voinovich, 2019). The latter study showed that the parthenogenetic laboratory strain of Trichogramma telengai Sor. harbored substantial variation between females in the percentage of diapause in their progeny. The variation in diapause incidence was statistically significant in most replicates of the experiment, including the cases where the total fraction of diapausing individuals approached 90%. The distribution of females by the percentage of diapausing progeny was always unimodal, indicative of a quantitative (not qualitative), or continuous, type of individual variation. There was a significant positive correlation between diapause incidence values in the progeny of the same wasp female that consecutively parasitized different egg batches of the host species, and thus individual properties of the females explained a large portion of the variation observed. The nature of this variation, however, remained an open question. The present study aims at finding out whether this variation is genetic or due to nonhereditary factors.

The experimental design was quite simple: we studied individual variation in diapause incidence among the progeny over two consecutive generations of isofemale substrains isolated from the main laboratory strain. The presence of a positive correlation between the two generations of the same substrain would indicate that the differences between maternal females are mostly genetic, whereas the absence thereof would suggest that the observed variation is due to nonhereditary processes.

MATERIALS AND METHODS

We used the same parthenogenetic laboratory strain of T. telengai as in the previous work (Reznik and Voinovich, 2019). Prior to the experiments, wasps had been reared on eggs of the grain moth Sitotroga cerealella (Oliv.) (Lepidoptera, Gelechiidae) under constant laboratory conditions (20°C, 18 h of light) for many years.

Each experimental replicate started with grain moth eggs being pasted with polyvinyl acetate glue onto cardboard cards and presented for 24 h to laboratory strain wasps for oviposition. Immediately following this, each card with parasitized host eggs was divided into two halves, which were individually incubated in large tubes (10 cm in length, about 3 cm in diameter) under a photoperiod of either 12 : 12 or 18 : 6 (light : darkness, h). The temperature in both photoperiodic regimens was the same at 20°C. On the day of mass emergence of adult wasps of this (first maternal) generation, females were individually allocated to small (5 cm in length, about 0.8 cm in diameter) numbered tubes with 50% honey solution applied to the walls as a source of carbohydrates. The progeny of each female from this first generation was considered as representing a substrain. Immediately after allocation to the individual tubes, the females of the first maternal generation were presented with numbered cards that contained 30–40 grain moth eggs. After 2 h, the females were returned to the photoperiodic regimens they had developed in and the cards with parasitized eggs (the first daughterly generation) were transferred to moderately diapause-inducing conditions (darkness, 14°C). After 2 days, females of the first maternal generation were presented for 24 h with numbered cards that contained the second portion of grain moth eggs (about 50 eggs per card). The cards with parasitized eggs (the second maternal generation) were transferred to individual tubes of the same size and incubated in the same regimen as the previous generation (either 12-h or 18-h light at 20°C).

On the day of mass emergence of adult wasps of the second maternal generation, females were kept in the same tubes they had developed in (depending on the maternal fecundity, the number of wasps per tube usually varied from 5 to 15 ind.) and presented for 2 h with numbered cards that contained 400–600 grain moth eggs. The cards with parasitized eggs (the second daughterly generation) were transferred to moderately diapause-inducing conditions (darkness, 14°C).

In order to accelerate development and adult emergence in nondiapausing individuals, 30 days after parasitization, cards with wasps of the first and second daughterly generations were transferred to environmental chambers set to 20°C and an 18-h day length. After 15–20 d (when the majority of actively developing individuals would have emerged), all the parasitized eggs were dissected to count the number of actively developing (adults and pupae) and diapausing (prepupae) progeny. The number of adults emerged was estimated based on the number of empty eggshells with emergence holes. Larvae that died at different developmental stages were dismissed. The first and second daughterly generation cards that had less than 5 survivors (either diapausing or active) were discarded. The final data set only contained the substrains for which diapause incidence was known in both consecutive generations. Thus, in 8 experimental replicates, we were able to collect data on the progeny of two consecutive generations of 428 substrains of T. telengai. It should be noted that in the first maternal generation, each substrain was represented by a single female and in the second maternal generation, by several females. The counts of actively developing and diapausing wasps in the first and second daughterly generations yielded a total of 24,069 ind. The separate sample sizes for the different replicates and photoperiodic regimens are summarized in Fig. 1 and Fig. 2.

The experimental replicates were never synchronous and, as a rule, contained individuals from different generations of the laboratory culture. Previous studies showed that the percentage of diapausing individuals varied noticeably from generation to generation in different strains and species of Trichogramma wasps, even under constant conditions (Zaslavski and Umarova, 1981; Voinovich et al., 2013; Reznik et al., 2015). Therefore, data from different replicates were analyzed separately and independently of each other. Significance of individual variation was tested with a chi-square test. Correlations were analyzed with ordinary linear regression. All analyses were performed in SYSTAT 10.2.

RESULTS AND DISCUSSION

In the vast majority of experimental replicates under short-day conditions (Fig. 1) and in all of the long-day replicates (Fig. 2), there was statistically significant individual variation in diapause incidence between the progeny of T. telengai females in the first and second maternal generations. Furthermore, as can be seen in the figures, long-day conditions resulted not only in higher significance values but also in a broader variation range. These findings confirm the results of our previous study (Reznik and Voinovich, 2019) where the range of individual variation also depended on the photoperiod and temperature experienced by the maternal and daughterly wasp generations. In nature, greater individual variance in the tendency to enter diapause under long-day conditions may contribute to a risk-spreading strategy whereby, under short-day photoperiods of autumn, before the onset of winter, diapause is induced in all or at least in the vast majority of wasps, but some individuals tend to enter diapause under long-day conditions, i.e., in summer, when average climatic variables still permit successful completion of one or more additional generations. Thus, the population has a greater chance of survival in case of early cold snaps or other extreme deviations from the climatic norm. Such individual variation of seasonal cycles was discovered, e.g., in the ragweed leaf beetle Zygogramma suturalis (F.) (Coleoptera, Chrysomelidae) (Vinogradova, 1988) and in many other insect species (Hopper, 1999; Joschinski and Bonte, 2021).

Fig. 1.
figure 1

Individual variation in diapause incidence among the progeny of short-day (12 h) reared Trichogramma telengai Sor. females. F1 and F2 columns: abscissa – percentage of diapausing progeny, mid-class values are shown; ordinate – distribution of females of different classes according to diapause incidence in their progeny. F1 – first generation of substrains (n1 – number of maternal females, n2 – number of daughters studied, p – significance of differences between the females according to chi-squared test). F2 – second generation of substrains (n1 – number of substrains, n2 – number of daughters studied, p – significance of differences between the substrains according to chi-squared test). F1–F2 column: abscissa – percentage of diapausing females of the first generation; ordinate – percentage of diapausing females of the second generation of the same substrains (r – correlation coefficient, n – number of substrains, p – significance of correlation), each symbol corresponds to one substrain, the line illustrates the linear regression equation. Rows A–H – experimental replicates.

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The results of our experiment suggest that the variation observed is mostly nonhereditary as the correlation between the diapause incidence values in two consecutive generations of the same substrains was nonsignificant in the vast majority of cases (Fig. 1 and Fig. 2). However, in two out of eight long-day replicates, where variation was more pronounced, the correlation was positive and significant (Fig. 2, replicate A) or even highly significant (Fig. 2, replicate C). Apparently, hereditary factors do contribute to the individual variation in the percentage of diapausing progeny between wasp females, but these hereditary differences can only be detected in the absence of substantially stronger confounding extrinsic factors, which were uncontrolled in our study. The nature of these factors remains obscure. The main abiotic factors that may have influenced the induction of diapause were controlled precisely: day length was set accurate to a few minutes, and temperature deviated from the set value by no more than 0.2–0.3°C. Furthermore, these deviations in conditions were the same for all of the individuals in a replicate and may have shifted the mean values or create differences between replicates but could not be responsible for differences between individuals and substrains, which were compared only within replicates.

Fig. 2.
figure 2

Individual variation in diapause incidence among the progeny of long-day (18 h) reared Trichogramma telengai Sor. females. Designations as in Fig. 1.

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As mentioned above, many insect species have been shown to exhibit interpopulational (in nature) and interstrain (in the laboratory) variation in the tendency to enter diapause (Danilevskii, 1961; Zaslavski, 1984; Tauber et al., 1986; Saulich, 1999; Denlinger, 2002; Saunders et al., 2002; Saulich and Volkovich, 2004; Danks, 2007; Tougeron, 2019; Saunders, 2020). In nature, differences between populations usually correlate with local dynamics of the main environmental factors (temperature, availability of food, etc.) and often show clinal patterns. Individual (intrapopulational and intrastrain) variation of diapause responses has been more rarely studied but, without doubt, is similarly widespread, as accumulation of interpopulational differences would not be possible in the absence of individual variation. It should be noted that the studies cited above mostly deal with natural populations or laboratory strains of bisexual insect species, while our work addresses a parthenogenetic strain where one would expect to find lower individual variation. Even in this respect, however, T. telengai is by no means an exception. For example, the pea aphid Acyrthosiphon pisum (Harris) (Hemiptera, Aphididae) was shown to have not only interclonal but also intraclonal variation in photoperiodic reactions controlling its seasonal cycle (Erlykova, 2003).

In nature, individual variation plays a beneficial role in insects as it increases the plasticity, evolutionary potential, and environmental robustness of their populations. By contrast, intrastrain variation of biocontrol agents not only provides material for selection but often appears to be a significant negative factor that hampers standardization of the reared stock, which must be taken into account in the development of industrial and laboratory rearing methods (Hoy, 1986; Hopper, 1999; Wajnberg, 2004; Routray et al., 2016; Bielza et al., 2020; Leung et al., 2020; Joschinski and Bonte, 2021).