Development and diapause induction of the Indian meal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) at different photoperiods

Diapause concerns the fascinating phenomenon in the biology of insect development which allows better understanding the local adaptation and phenotypic plasticity to seasonal variations in environment. There is lot of reasons to carry out the research on diapause both for fundamental and applied sciences. Photoperiod is one of the main environmental cues followed by insects to predict the forthcoming seasonal changes and to adapt these changes in their life-history traits. Thus, the effect of different photoperiod regimes on development and diapause induction of larvae of the Indian meal moth Plodia interpunctella (Hübner) was evaluated at a constant temperature of 17 °C. Development was significantly faster at a photoperiod of 12:12 light:darkness (L:D) than at 8:16, 10:14, 14:10 and 16:8 L:D. A photoperiod of 12:12 (L:D) induced most larvae (≥ 71%) to enter diapause, while this percentage was slightly lower (60%) at both shorter(8 h) and longer (16 h) day lengths (50%). The different photoperiod regimes did not affect the percentage of adult emergence. Fat and protein composition of the diapausing larvae differed significantly among treatments as well as between diapausing and non-diapausing larvae. Larvae developing from 8:16 (L:D) contained the maximum amount of protein (36.8%) compared to other regimes, while the minimum amount (21.0%) was noted in larvae that developed at 16:8 (L:D). Six types of fatty acids were detected in the larvae: myristic acid (methyl tetradecenoate), palmitoleic acid (9-hexadecenoic acid, methyl ester), palmitic acid (hexadecenoic acid, methyl ester), linoleic acid (9, 12-Octadecadienoic acid (Z, Z), methyl ester), oleic acid [9-octadecenoic acid, methyl ester (E)] and stearic acid (octadecanoic acid, methyl ester). The results also reveal that the percent of fatty acids detected in the diapausing larvae varies significantly and the same trends imply in the interaction of fatty acid and photoperiod regimes. Moreover, three quarters of the total variance was accounted for by the Principal Component Analysis (PCA) of the fatty acids. Different proportions of fatty acids were noted among treatments, suggesting that photoperiod influences a number of key biological traits in P. interpunctella, much more than the percentage of the diapausing larvae per se.

www.nature.com/scientificreports/ during their diapause, similar to the presumed role of heat-shock or stress proteins. In contrast, DAP is produced in advance of the environmental stress, unlike heat shock proteins which are synthesized in response to a stress stimulus 53 . Plodia interpunctella refers as one of the most cold hardy stored product pests 55 and the diapausing larvae may spend six or more months in diapause 18,22 . The low temperature enhanced the response in diapause at short-day conditions. For example, the maximum (80%) diapause response was observed in flesh flies when reared at 25 °C while the diapause incidence is elevated to nearly 100% at 18 °C. In the present study, diapause induction of P. interpunctella was systematically investigated under laboratory conditions, at a constant low temperature level (17 °C), for which there are no data available, despite the fact that exposure of P. interpunctella to low temperatures can be further implemented for mass rearing of parasitoids 56 . This was carried out at different photoperiod regimes. In addition to diapause induction, we examined larval development, as well as the abundance of proteins and fatty acids in the larvae.
Fatty acid profiles. Results clearly revealed that there was a significant (F = 29.61; df = 11,30; P < 0.001) impact of photoperiodic response on the fatty acid profiles in diapausing P. interpunctella larvae (Table 1). We detected six types of fatty acids, i.e. myristic acid (methyl tetradecenoate), palmitoleic acid (9-hexadecenoic acid, methyl ester), palmitic acid (hexadecenoic acid, methyl ester), linoleic acid (9, 12-octadecadienoic acid (Z, Z), methyl ester), oleic acid [9-octadecenoic acid, methyl ester (E)], and stearic acid (octadecanoic acid, methyl ester) ( Table 1)    Fatty acid composition and ratios in diapausing larvae are summarized in Table 1 and Fig. 7. Approx. three quarters of the total variance was accounted for by PCA of the seven fatty acids. First principal component (PC) has large positive associations with 9-hexadecenoic and 9,12-octadecadienoic (Z,Z). The PCA biplot shows both Figure 6. Percentage (% ± SE) of total protein in P. interpunctella larvae reared at 17 °C and different photoperiod regimes (means followed by the same letter are not significantly different; HSD test at 5%). Table 1. Percent of fatty acids in non-diapausing (ND) and diapausing P. interpunctella larvae that had been exposed to different photoperiod regimes at 17 °C. -Not available.   www.nature.com/scientificreports/ PC scores of samples (dots) and loadings of variables (vectors) (Fig. 8). The PCA analyses indicated that the fatty acids octadecadienoic and methyl tetradecanoate exhibited positive correlation while they were negatively correlated with all other fatty acids. Moreover, most of the fatty acids including hexadecanoic acid, 9,12-octadecadienoic (Z,Z)-,9-octadecenoic acid, 9-hexadecenoic acid and methyl stearate showed positive correlation among them except 9-octadecenoic acid and methyl stearate. The biplot indicates that the photoperiods 8:16 and 10:14 (L:D) have more or less similar response patterns over variables compared to other photoperiods (Fig. 8).

Discussion
Previous research indicated that a combination of short photophases with low temperatures can cause a rapid diapause induction of P. interpunctella larvae 16,17 . In general, photophases that were 13 h or shorter cause a quick diapause induction 19 , but diapause may not occur if the prevailing temperatures are high 19,57 . However, it has not been clarified whether the reduced activity of this species during the cold period of the year is related to increased diapause or decreased mobility due to reduced metabolism, although these two are likely related 11,58 . For P. interpunctella larvae, diapause is prevented at an increase of photoperiod to 16:8 (L:D), suggesting that temperature is less important than photoperiod in determining diapause 18 . Our results partially stand in accordance with the above observations, as the highest percentage of diapause induction was recorded at 12:12 (L:D). However, no significant differences were noted among the photoperiod levels tested here, indicating that the photophases evaluated were perhaps too narrow to detect differences that may have been observed if more photophases had been tested. Wang et al. 59 systematically investigated the diapause induction and termination of small brown planthopper, Laodelphax striatellus (Fallén) by changing photoperiod and temperature. They also concluded that the temperatures ranging from 18 to 28 °C greatly influenced the incidence of diapause in L. striatellus. For instance, the photoperiodic response curve at 20 °C showed a gradual decline in diapause incidence in ultralong nights, and continuous darkness resulted in 100% development. The different photoperiod regimes that were used in our experiments, in conjunction with exposure to 17 °C, might have affected diapause induction differently, as compared with the results reported by Bell 18 , but the diapause attributes of the different populations may account for the differences noted from other studies. Interestingly, we have shown little response to photoperiod with our P. interpunctella population. Still, we have noted different patterns of adult eclosion and sex ratio as a result of photoperiod, suggesting that different photoperiodic regimes may affect progeny production capacity of the exposed individuals. Larval weight was notably increased at certain photoperiods. At the same time, at these photoperiods, the fatty acids composition was different, as compared with 8:16 and 14:10 (L:D). Differences in fatty acids among individuals that had been exposed to various photoperiods were previously noted for other species as well. For instance, for the silk moth, Bombyx mori (L.), Shimizu 60 found that phosphatidylcholine of diapausing eggs contained more linolenic acid and less myristic acid than that of non-diapausing eggs. Similar reports have illustrated considerable differences between diapausing and non-diapausing individuals of the pink bollworm, Pectinophora gossypiella (Saunders), a feature linked to the timing of diapause termination 61,62 . The actual effect of the fatty acid composition on P. interpunctella larvae in diapause induction and termination is poorly understood, and requires additional investigation.
In a study of the tobacco moth, Ephestia elutella (Hübner), Bell 63 underlined the importance of continuous rearing conditions in the laboratory in relation to diapause induction, which may not occur in "real world" www.nature.com/scientificreports/ conditions, in terms of regulation of biological cycle and succession of the generations. In this context, it has been reported that the diapause has been a major roadblock to developing control programs for many pests 64 . For instance, the sterile insect technique (SIT) and augmentative natural enemy control have been neither practical nor possible due to diapause responses that prevent or interfere with continuous mass rearing 64 . There are many examples in this regard, including the European cherry fruit fly 65 , the Apple maggot fly 66 , the Chinese citrus fruitfly 67 , the Russian melon fly 68 , and processionary moths 69 . Bloemi et al. 64 noted that diapause induction, originally developed for individually reared codling moth, can be applied to the mass-rearing system used by the Sterile Insect Release (SIR) eradication program. The current research also suggests that there are some approaches that can potentially disrupt diapause and facilitate mass rearing. In this context, generalizations regarding the "optimum" conditions that are related with diapause should be avoided, as any "diapause"-related control strategy may not be accurate. On the other hand, considering that P. interpunctella is an ideal species to rear insect parasitoids, standardization of life table characteristics of a given population may be valuable to produce large numbers of parasitoids whenever these are needed 70,71 . Hence, considering our results, we have found that larvae of P. interpunctella can be used with success at various conditions, as the effect of different photoperiod regimes does not drastically affect diapause induction, when these larvae are reared at 17 °C. As Bell 18 showed that diapause induction at a photophase of ≤ 13 h was not different between 20 and 25 °C, our study shows that pre-exposure at 17 °C might have widened the photoperiodic requirements towards this direction. Generally, the diapause of this species is classified as a long-day type 23,24 , while experiments with different photoperiodic regimes did not show a significant effect on diapause, unless the photoperiod was combined with specific thermo-period regimes 23,24,72 . It is well established that diapause in P. interpunctella is mostly related to scotophase rather than photophase, as constant scotophases provided similar diapause induction even when photophases are disrupted 73 . In this context, the critical scotophase varies from 8 to 12 h even when the photophase is kept at a 24-h duration 72 . This partially explains our results, considering that the scotophase used here was within these limitations.

conclusions
In summary, the results of the present study show specific photoperiodic associations in P. interpunctella larvae that seem to perform similarly across a wide range of photoperiods, provided that these individuals are exposed to 17 °C. Cold hardiness may be directly related to diapause induction 73,74 . As this temperature does not cause high larval mortality in this species 11,12,73 , it should be considered further for parasitoid rearings in mass production protocols. Diapause induction of P. interpunctella larvae. Neonate larvae (1d old) of P. interpunctella were transferred individually into transparent plastic rearing trays (9.6″L × 4.1″W × 2.0″H) (HL-B025, Jiangsu, China) containing fifty small holes (2 ml) filled with food medium (5 g) at 17 °C and 60% r.h. Trays were covered with a transparent plastic sheet with tiny holes to allow exchange of air. Five photoperiods, i.e. 8:16, 10:14, 12:12, 14:10 and 16:18 (L:D) were tested for larval diapause induction. The experimental set up for photoperiodic diapause induction is presented in the schematic diagram of Fig. 9. The development of larvae was observed daily during the photophase. If a larva did not pupate after being reared for 40 d at 17 °C, it was considered to be in diapause. Diapausing larvae were also identified based on their extended larval developmental period, large size and yellowish color due to accumulated fat 19,21 . The number of larvae, pupae and moths per tray were recorded separately for each photoperiod regime and the percentage of diapausing larvae was then calculated. Mature larvae reared under each photoperiod regime were weighed using an electronic balance (FA-N/A-N, Shijiazhuang, China). Longevity, fecundity and eclosion of adults developing from diapausing and non-diapausing larvae were also recorded. The critical photoperiod is defined as the photoperiod that induces 50% of the maximum inci- www.nature.com/scientificreports/ dence of diapause 1 . There were three replications containing 50 larvae for each photoperiod regime. Under these conditions (17 °C and 60% r.h.), non-diapausing larvae pupated in the food and emerged as adults by day 60 after oviposition. As a light source, two 10-W daylight fluorescent bulbs were used and the photoperiodic cycles were controlled by a 24-h time switch 18 . The scotophase was controlled manually by wrapping cardboard boxes containing experimental material in a black polythene sheet.

Methods
protein analysis. Proteins in non-diapausing and diapausing mature larvae of P. interpunctella were measured according to Kjeldal method 76 . Percentages of nitrogen were transformed into protein content by multiplying a conversion factor of 5.3 as suggested 77,78 . There were three replicates, each with ten larvae for each of the photoperiod regimes.
Fatty acid extraction and analysis. Lipids were extracted according to the method suggested by Folch et al. 79 . Twenty-five non-diapausing and diapausing mature larvae of P. interpunctella were homogenized in glass tubes and extracted three times with chloroform:methanol 2:1 (v/v). Supernatants were pooled and mixed with aqueous 0.88% KCl. After final centrifugation, the lower phase was collected and dried under a stream of N 2 . The lipid samples were processed for fatty acid analyses following methods described by Metcalf et al. 80 . The fatty acids of isolated lipids were methylated into reaction vials by refluxing with sodium methoxide (2%) for 10 min at 100 °C and then were transmethylated by refluxing with 2.175 ml boron trifluoride methanol 14% for 3 min at 100 °C. The fatty acid methyl esters (FAMEs) were extracted from the reaction vials three times with hexane, and concentrated. The fatty acids (FA) in the total lipids were determined as methyl ester derivatives by gas chromatography employing standard protocols 81 . The FAMEs were analyzed on a GCMS-QP2020 (SHIMADZU Co., Kyoto, Japan). The split ratio was 50:1 and 1 μl of solution was injected into the column. Helium was used as the carrier gas with flow rate of 1 ml/min. The oven temperature was kept at 140 °C for 5 min, increased at a rate of 3 °C/min to 240 °C, and held at 240 °C for 10 min. The injector and detector temperatures were maintained at 260 °C. The fatty acids were identified by comparing their retention times with those of the FAME standards under the same conditions. The fatty acid analyses were performed at Bangladesh Council of Scientific and Industrial Research (BCSIR) Laboratories, Rajshahi, Bangladesh.
Statistical analysis. Assumptions of normality and homogeneity of variance were tested using Levene's 82 method and indicated that the data should be arcsine transformed before the analysis. Then, the data were analyzed through ANOVA using the PROC GLMMIX (SAS Version 9.2, 2008) 83 , separately, for each of the scenarios indicated above. Means were compared by Tukey-Kramer HSD test at 5%. Untransformed means and standard errors are reported to simplify interpretation. Multivariate statistical procedures including principal component analysis (PCA) was employed to assess the differentiation as well as the relationship between fatty acids and photoperiod regimes. The PCA biplot was also designed to take multidimensional data sets and reduce their dimensions by determining one or more linear combinations of the variables. Moreover, PCA biplot showed both PC scores (PCs) of photoperiod regime (dots) and loadings of percent fatty acids (vectors). The PCs were sufficient to describe the essence of our data since the scree plot showed an ideal curve.