The Effect of Combinations of Food Insects for Continuous Rearing of the Wing Polymorphic Water Strider Limnogonus Fossarum Fossarum (Hemiptera: Gerridae)

Journal of Insect Science (2016) 16(1): 80; 1–7 doi: 10.1093/jisesa/iew059 Research article

The Effect of Combinations of Food Insects for Continuous Rearing of the Wing Polymorphic Water Strider Limnogonus Fossarum fossarum (Hemiptera: Gerridae)

Y. Hirooka, C. Hagizuka, and I. Ohshima1

Department of Life and Environmental Sciences, Kyoto Prefectural University, Sakyo, Kyoto, 606-8522 Japan ([email protected]; [email protected]; [email protected]), and 1Corresponding author, e-mail: [email protected] Subject Editor: Philippe Usseglio-Polatera

Received 12 February 2016; Accepted 16 June 2016

Abstract The water strider Limnogonus fossarum fossarum (F.) (Hemiptera: Gerridae) shows a macropterous, micropterous, and apterous polymorphism. Although a long photoperiod condition induces winged morphs, preliminary studies have revealed that crossing between winged morphs increased the proportion of macropterous individuals, suggesting that the genetic factors also affect wing-morph determination in this species. Assessing the genetic backgrounds of wing polymorphism requires constant and repeatable methods for rearing. This study attempts to establish a continuous rearing method for L. f. fossarum under constant diet conditions. Initially, we maintain the water striders with two Drosophila species as a food, but viability until adulthood is less than 20%. We then add the storage pest Plodia interpunctella (Hu¨ bner), which are readily reared in the laboratory, to the diets. As a result, nymphs fed on P. interpunctella (even only until the second instar) show signiﬁcantly higher viability and shorter developmental period than nymphs fed on Drosophila alone. Moreover, feeding on D. melanogaster (Meigen) reared on cholesterol-enriched medium instead of a normal medium signiﬁcantly increases viability in the next generation. This means that only the two food-insect species are enough for establishing a substantial number of individuals in segregating generations (F2 and backcross), limiting DNA and RNA con- taminations from food insects with genome information. Thus, the present rearing method opens the way to elucidating the genetic backgrounds of the wing polymorphism in L. f. fossarum.

Key words: Drosophila melanogaster, Plodia interpunctella, cholesterol

Wing morphologies have diverged greatly among the insect species. Nishimoto 2007] and interspecific interactions [Dixon and In the course of the evolution of insects, some species have lost their Agarwala 1999; Sloggett and Weisser 2002; Kunert and Weisser wings secondarily, and others show wing polymorphisms within 2003]), and abiotic (e.g. photoperiod [Harada and Taneda 1989; species (Roff 1990). Wing polymorphisms in insects generally affect Harada and Numata 1993; Inoue and Harada 1997; Harada their flight ability, resulting in differences in dispersal abilities 1998a; Harada et al. 2005; Harada et al. 2011], temperature among individuals with different wing morphs (Harrison 1980). [Harada et al. 2003], salinity [Kishi et al. 2007], and dryness Thus, investigating the mechanisms controlling wing polymorphism [Harada 1998b; Kishi et al. 2002]) environmental cues affect wing- is crucial for understanding the evolution of life history (Zera and morph determination (Braendle et al. 2006; Simpson et al. 2011). Denno 1997). In contrast, several studies have shown that genetic factors contrib- Both environmental and genetical factors influence the deter- ute to wing-morph determination in water striders and plant hop- mination of wing morphs in insects (Roff 1986; Simpson et al. pers as well as aphids (Zera et al. 1983; Mori and Nakasuji 1990; 2011). Environmentally determined wing polymorphisms are Matsumura 1996; Caillaud et al. 2002). Thus the genetic basis and known as wing polyphenisms in which the same genotype produces maintenance mechanisms of genetically determined wing poly- discontinuous phenotypes (Braendle et al. 2006). Much study on morphisms have received ongoing scrutiny (Harrison 1980; Roff wing polyphenism has been done using aphids and water striders 1986; Zera and Denno 1997). However, we know little about the and revealed that both biotic (e.g. density [Harada et al. 1997; genetic backgrounds underlying genetically determined wing Harada and Spence 2000], nutrient conditions [Harada and polymorphisms.

VC The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Journal of Insect Science, 2016, Vol. 16, No. 1

The water strider Limnogonus fossarum (Hemiptera: Gerridae) photoperiod in the laboratory. We maintained two wild types is a tropical and subtropical species in the Old World, and the sub- (Oregon-R and Canton-S) and two mutants, Curly (Cy) and vestigial species L. f. fossarum is distributed in the Oriental region (Andersen (vg)ofD. melanogaster, and randomly used these as food for L. f. 1975). L. f. fossarum shows wing polymorphism that produces mac- fossarum. In addition to the two Drosophila species, P. interpunc- ropterous, micropterous, and apterous adults (Andersen 1982), and tella were collected near the campus of Kyoto Prefectural University these morphs are easily distinguished by eye. Since L. f. fossarum (Shimogamo, Sakyo, Kyoto, Japan). Adult pairs of P. interpunctella does not have any dormancy periods in its life history and its gener- were introduced to single clear plastic containers (135 85 ation time is relatively short (ca. 50 days, see Results section), more 30 mm) containing brown rice as larval food and maintained at than five generations can be continuously reared in the laboratory 28 C under a 16:8 (LD) h photoperiod in the laboratory. Emerged per year. Therefore, L. f. fossarum could be a novel model system adults of all these food insects were killed by freezer and used as for studying what extent genetic and environmental factors influ- prey in experiments of food insect combinations. ence the determination of wing morphs. General rearing method: Eggs from each collected wild female or In this study, our goal is to establish a method for rearing L. f. the laboratory population were incubated on a water-soaked filter fossarum successively in the laboratory under constant environmen- paper in plastic Petri dishes (52 mm diameter, 13 mm depth). Newly tal conditions. Because environmental conditions influence wing- hatched nymphs were introduced to translucent plastic containers morph determination (Braendle et al. 2006; Simpson et al. 2011), es- (60 mm 60 mm 45 mm) in groups of up to five individuals from tablishing uniform environmental conditions for rearing is very im- the same parent. These nymphs were then transferred individually to portant for distinguishing genetic contributions from environmental other clear plastic containers (60 mm 60 mm 95 mm; Plant Box, contributions to wing-morph determination. Of the biotic and abi- #CUL-JAR300, AGC Techno Glass Co., Ltd., Tokyo, Japan) at the otic conditions affecting wing-morph determination (Braendle et al. third or fourth instar depending on the subsequent experimental de- 2006; Simpson et al. 2011), nutrients are the most difficult condition sign. Each container was filled with 5 mm water that had been dech- to establish uniformly in water striders because rearing with only a lorinated by being incubated over 24 h in the laboratory. A cut piece single food-insect species often results in very low survival rates in of extruded polystyrene plate was added on the water surface as a resting site. All food insects, polystyrene plates, and water in the their nymphal stage (for reviews, see Lefcheck et al. 2013; De Clercq rearing containers were replaced daily regardless of what food in- et al. 2014). For this reason, we have to use multiple insect species sects were provided to the water striders. Eggs and nymphs were to recover high survival rates in successive rearing of water striders, maintained at 25 C under a 16:8 (LD) h photoperiod in all but this increases the complexity of contaminating DNA and RNA experiments. from the food-insect species and violates the trimming of the con- Food insect combinations: To assess effective food insect com- taminating sequences in the step of genome-data analyses. Thus, binations for rearing L. f. fossarum successively, we estimated via- food insect species should be those which genome information is bility from F eggs to adults, fertility of F adults (i.e. germination available, and we should establish a method using as few food spe- 1 1 rates of F eggs), hatchability of F nymphs, and viability of F cies as possible. 2 2 2 nymphs until the second instar or adults with the following four Here, we present a method for successive rearing of L. f. fossa- food insect experiments. Detailed names of food insect species and rum using two food insect species, the fruit fly Drosophila mela- the amount of each food insect used per day in each experiment are nogaster (Diptera) and the Indian meal moth Plodia interpunctella shown in Table 1. (Lepidoptera), whose genome information is available (Adams et al. Experiment 1: D. melanogaster and D. hydei. F1 offspring 2000; Harrison et al. 2012). The two food species are readily main- derived from 26 females from the spring samples (collected on 3 tained in the laboratory and have short generation times. Our April 2012) were used in this experiment. Hatched F1 nymphs were method uses a cholesterol-enriched medium for rearing D. mela- fed on two species of Drosophila (Dm þ Dh treatment, Table 1). F1 nogaster. This significantly reduces mortality of its predator, L. f. viability was estimated as the percentage survival of individuals until fossarum, and enables us to rear L. f. fossarum successively with adulthood out of all eggs obtained. F1 offspring which successfully only two food-insect species. Thus, our present rearing method survived to adulthood were crossed with other adults from the same opens the way to make L. f. fossarum a suitable system for studying parent (i.e., full-sib mating) and F2 eggs were obtained. Eyespots the genetic mechanisms underlying wing polymorphisms. emerge 4 d after oviposition when eggs have been successfully ger- minated. Thus, we checked for the presence of eyespots on each egg Materials and Methods 6 d after oviposition, and the presence of eyespots was regarded as an indicator of successful egg germination. Insects: Adults of L. f. fossarum (P generation) were collected from Experiment 2: P. interpunctella for 1st and 2nd instars in add- Ishigaki Island (2422-270N, 1249-120E), Okinawa Prefecture, ition to D. melanogaster for all instars. F1 offspring from 16 females Ryukyu Archipelago, Japan. Twenty-six females and 16 females from the summer samples (collected on 13 August 2012) were used were obtained on 3 April and 13 August 2012, respectively. for this experiment. To assess the effect of adding P. interpunctella Collected adult females were maintained with D. melanogaster as as a food insect, 20 eggs each from 16 females were split 1:1 into food and allowed to lay eggs at 25 C under a 16:8 (L:D) h photo- two food treatments. In the first treatment (Dm treatment), L. f. fos- period in the laboratory of Kyoto Prefectural University, and ob- sarum nymphs were fed only on D. melanogaster for all stages; in tained eggs were used for the subsequent experiments. We also the second treatment (Dm þ Pi1, 2 treatment), the nymphs were fed established a laboratory population from two females sampled in daily, alternating between D. melanogaster and P. interpunctella, spring and 16 females sampled in summer 2012, and used these for until the end of the second instar and then fed only on D. mela- the experiments. nogaster until adulthood (Table 1). In each treatment, we estimated

As food insects, two species of Drosophila (D. melanogaster and F1 viability as the mean percentage of individuals surviving until D. hydei Sturtevant) were maintained on a medium Jazz-mix (Fisher each instar or adulthood of 16 sibling groups and compared the via- Scientific Inc., Tokyo, Japan) at 25 C under a 16:8 (LD) h bilities of the two treatments in each stage. We also compared F1 Journal of Insect Science, 2016, Vol. 16, No. 1 3

Table 1. Species and amount of adult food insects fed to each stage of L. f. fossarum

Rearing methods 1st 2nd 3rd 4th 5th Adult

Experiment 1 DmþDh Dm 1 Dm 2 Dm 3 Dh 1 Dh 2 Dh 3 Experiment 2 Dm Dm 1 Dm 2 Dm 2 Dm 3 Dm 3 Dm 4

DmþPi1, 2 Dm 1, Pi 1/3 Dm 2, Pi 1/2 Dm 2 Dm 3 Dm 3 Dm 4 Experiment 3 DmþPi1, 2, 3 Dm 1, Pi 1/3 Dm 2, Pi 1/2 Dm 2, Pi 1 Dm 3 Dm 3 Dm 4 DmþPi1, 2, 5 Dm 1, Pi 1/3 Dm 2, Pi 1/2 Dm 2 Dm 3 Dm 3, Pi 1 Dm 4

Experiment 4 DmþPi1, 2, 5 Dm 1, Pi 1/3 Dm 2, Pi 1/2 Dm 2 Dm 3 Dm 3, Pi 1 Dm 4 DmcþPi1, 2, 5 Dmc 1, Pi 1/3 Dmc 2, Pi 1/2 Dmc 2Dmc 3Dmc 3, Pi 1 Dmc 4

Dm, Dh, and Pi indicate Drosophila melanogaster, D. hydei, and Plodia interpunctella, respectively. Numbers after abbreviations indicate daily amounts of each food-insect species per L. f. fossarum larva. Under lines indicate periods in which L. f. fossarum were reared in groups.

hatchability, viability until the second instar, and viability until Table 2. Ingredients in the cholesterol-added medium for adulthood in the F2 generation), and each parameter was compared Drosophila melanogaster in the Experiment 4 (amounts per 100 between the Dm þ Pi and Dm þPi treatments. culture bottles) 1, 2, 5 C 1, 2, 5 Statistical analysis: We used Student’s t-test in all analyses for Material Amount split-design experiments in Experiments 2–4. However, we used

Welch’s t-test for the hatchability and viability of the F2 generation Medium (Jazz-mix)a 68 g in the Experiment 4 due to the unequal variances between the Cholesterolb 0.36 g Distilled water 340 ml Dm þ Pi1, 2, 5 and DmC þPi1, 2, 5 treatments. In every analysis, we used arcsine square-root-transformed the viability, fertility and a Fisher Scientiﬁc Inc., Illinois, USA. hatchability per sibling group in the F1 or F2 generation in order to b Wako Pure Chemical Industries, Ltd., Osaka, Japan. satisfy the requirement of normality. We also analyzed the effect of food-insect combinations on wing morphs (macropterous, microp- developmental periods from hatching to adult eclosion using F1 off- terous, and apterous) by using the ‘multinom’ function implemented spring that successfully became adults. We crossed F1 offspring from in the ‘nnet’ package (Ripley and Venables 2016) in Experiments 2– four randomly selected sibling groups and obtained 50 F2 generation 4 with six models (Supp Table S1 [online only]). Model fits were eggs from each group. We counted fertile eggs of the F2 generation evaluated using the Akaike Information Criterion (AIC). All statis- using the same method as in Experiment 1. We assessed F2 hatch- tical analyses were carried out using the R version 3.2.3 (R ability and viability until second instar only in the Dm þ Pi1, 2 treat- Developmental Core Team 2015). ment because all from the Dm treatment were sterile. Experiment 3: P. interpunctella for third or fifth instars in addition to the dietary conditions from Experiment 2. In this experi- Results ment, the laboratory population of L. f. fossarum was used. Six Experiment 1: D. melanogaster and D. hydei: Of 658 F1 eggs, only females that had started oviposition (P generation) were isolated in- 102 individuals survived until adulthood (viability 15.5%). dividually into Plant Boxes and were allowed to lay eggs to produce Although 1218 F2 eggs were laid by 14 F1 females, there were no fer-

F1 offspring. To assess to which instars it would be most effective to tile eggs under these food conditions. Thus, we could not assess the provide P. interpunctella as food, 20 F1 eggs each from six females hatchability and viability of the F2 generation, suggesting that we were split 1:1 into the Dm þ Pi1, 2, 3 treatment and Dm þ Pi1, 2, 5 could not rear L. f. fossarum continuously using only Drosophila treatment (Table 1). In the F1 generation, we recorded viability until species as food insects. adulthood from eggs, length of developmental period from first in- Experiment 2: P. interpunctella for 1st and 2nd instars in add- star to adult, and fertility of F1 adults. In the F2 generation, for each ition to D. melanogaster for all instars: In the Dm þ Pi1, 2 treatment, treatment, we recorded nymph hatchability and viability until the se- over 70% of individuals successfully survived to adulthood (Fig. 1). cond instar in the same manner as in Experiment 2. Each parameter In the Dm treatment, on the other hand, viability decreased suddenly was compared between the Dm þ Pi1, 2, 3 and Dm þ Pi1, 2, 5 treat- after the fourth instar mainly due to failure to molt, and in 10 of 16 ments. We only assessed F2 viability until adulthood in the progeny sibling groups, no individuals survived until adulthood. Nymphal of the Dm þ Pi1, 2, 5 treatment due to the very low survivorship of viability was significantly higher in the Dm þ Pi1, 2 treatment than in nymphs from the Dm þ Pi1, 2, 3 treatment (see Results). the Dm treatment starting in the fourth instar (Fig. 1; until second Experiment 4: D. melanogaster reared on cholesterol-added me- instar t ¼ 0.206, P ¼ 0.839; until third instar t ¼ 0.124, P ¼ 0.902; dium for all instars and P. interpunctella for first, second, and fifth until fourth instar t ¼ 2.33, P ¼ 0.0270; until fifth instar t ¼ 6.25, instars. This experiment also used the laboratory population of L. f. P ¼ 6.97e7; until adulthood t ¼ 11.8, p ¼ 8.52e13; df ¼ 30 for all fossarum and five females that had started oviposition (P gener- t-tests). ation) were isolated individually into Plant Boxes and were allowed The mean developmental period was 27.6 6 1.62 d (6 SD, to lay eggs to produce F1 offspring. To assess the effect of using D. N ¼ 17) in the Dm þ Pi1, 2 treatment, whereas it was 29.6 6 2.43 d melanogaster reared on a cholesterol-added medium (Table 2)asa (6 SD, N ¼ 7) in the Dm treatment; there was a significant differ- food insect, 20 F1 eggs each from five females were split 1:1 into the ence between the two treatments (t ¼2.35, P ¼ 0.0279, df ¼ 22,

Dm þ Pi1, 2, 5 treatment and DmC þPi1, 2, 5 treatment (Table 1). We t-test). recorded the same parameters as in Experiment 3 (viability until In the Dm þ Pi1, 2 treatment, although the mean fertility of F1 adulthood from eggs, length of developmental period from first in- adults was above 90%, in the subsequent F2 generation, mean star to adult, and fertility of adults in the F1 generation; nymph hatchability and viability until the second instar decreased to 4 Journal of Insect Science, 2016, Vol. 16, No. 1

n.s. n.s. *** n.s. n.s. n.s. n.s. n.s. n.s. *

d 100

o 100

t e d

s Dm+Pi1, 2, 5

o l

y Dm +Pi h

t c 1, 2, 5

e 50

Dm c e

50 a

e t

Dm+Pi n

1, 2 c

i v

r 0

u Fertility Hatchability Second Third Fourth Fifth Adult s

instar instar instar instar

n e

c Fig. 3. Comparison of the fertility and survival rate between F2 generations r

e 0

P Second Third Fourth Fifth Adult fed on normal D. melanogaster (Dm þ Pi1, 2, 5) and on D. melanogaster reared instar instar instar instar on the cholesterol-added medium (Dmc þPi1, 2, 5). Error bars are standard deviations. n.s. P > 0.05, * P < 0.05 (Welch’s t-test). Fig. 1. Comparison of survival rates between F1 generations fed on Plodia in

ﬁrst and second instars (Dm þ Pi1, 2) and fed only on Drosophila in all instars and adulthood (Dm). Error bars are standard deviations. n.s. P > 0.05, the second stadium in the Dm þ Pi1, 2, 3 treatment (Fig. 2). On the

* P < 0.05 (t-test). contrary, in the Dm þ Pi1, 2, 5 treatment, mean F2 hatchability and viability until the second instar were 89.3% and 84.7%, respectively (Fig. 2); there were significant differences in hatchability and viabil- n.s. * * ity between the two treatments (hatchability t ¼ 3.42, P ¼ 0.00653, 100 viability until second instar t ¼ 8.89, P ¼ 4.62e6, df ¼ 10, t-test).

The mean percentile viability until adulthood in the Dm þ Pi1, 2, 5

a r e 6 6

treatment was 53.0 23.6 ( SD, N ¼ 4).

s i

Experiment 4: D. melanogaster reared on cholesterol-added me-

e dium for all instars and P. interpunctella for first, second, and fifth

o instars: F viabilities until adulthood were not significantly differen- l y Dm+Pi 1

t 1, 2, 3 e

i 50

e t

r tiated between the two treatments (Dm Pi 70.0%,

d þ 1, 2, 5 ¼

e Dm+Pi1, 2, 5

e a

g DmC þPi1, 2, 5 ¼74.0%; t ¼ 0.410, P ¼ 0.693, df ¼ 8, t-test) as in

t e

n Experiment 3 (Supp Fig. 2 [online only]). The mean developmental

r e

P period was 28.6 6 0.49 d (6 SD, N ¼ 5) in the Dm þ Pi1, 2, 5 treat-

ment and 28.3 6 0.74 d (6 SD, N ¼ 5) in the DmC þPi1, 2, 3 treat- 0 Fertility Hatchability Successful eclosion ment; there was no significant difference between the two into second instar treatments (t ¼0.65, P ¼ 0.533, df ¼ 8, t-test). There was no significant difference in fertility between the two treatments (Fig. 3; Fig. 2. Comparison of the fertility and survival rate between F2 generations t ¼0.7244, P ¼ 0.490, df ¼ 8, t-test). produced by F1 offspring fed on Plodia in the ﬁrst through third instars

(Dm þ Pi1, 2, 3) and in the ﬁrst, second and ﬁfth instars (Dm þ Pi1, 2, 5). Error In F2 generation, hatchability and viability until each instar and bars are standard deviations. n.s. P > 0.05, * P < 0.05 (t-test). adulthood are consistently higher in the DmC þPi1, 2, 5 treatment

than in the Dm þ Pi1, 2, 5 treatment, although significant differences 53.9 6 40.5% and 0.52 6 1.04%, respectively (6 SD). In the Dm between the two treatments are detected in the viability until adult- treatment, no fertile eggs were obtained as in Experiment 1, hood alone (Fig. 3; hatchability t ¼1.003, P ¼ 0.346, df ¼ 7.825, although a single F1 female started oviposition. viability until second instar t ¼1.727, P ¼ 0.146, df ¼ 4.891; until Experiment 3: P. interpunctella for third or fifth instars in add- third instar t ¼1.357, P ¼ 0.243, df ¼ 4.185; until forth instar ition to the dietary conditions from Experiment 2. F1 viabilities until t ¼1.386, P ¼ 0.233, df ¼ 4.352; fifth instar t ¼2.353, adulthood were high (Dm þ Pi1, 2, 3 ¼80.0%, Dm þ Pi1, 2, p ¼ 0.0515, df ¼ 6.877; until adulthood t ¼2.484, p ¼ 0.0381,

5 ¼83.3%) in both treatments, and there were no significant differ- df ¼ 7.935; Welch’s t-test). There was no significant difference in the ences between the two treatments in viabilities until each instar mean larval developmental period between the DmC þPi1, 2, 5 treat-

(until second instar t ¼0.202, P ¼ 0.844; until third instar ment (42.4 6 2.54 d [6 SD, N ¼ 5]) and the Dm þ Pi1, 2, 5 treatment t ¼ 0.483, P ¼ 0.639; until fourth instar t ¼ 0.661, P ¼ 0.524; until (44.2 6 2.36 d [6 SD, N ¼ 3]) (t ¼ 1.00, p ¼ 0.355, df ¼ 6, t-test). fifth instar t ¼ 0.788, P ¼ 0.449; df ¼ 10 for all tests; t-test) as well Effect of food-insect combinations on wing morphs: Model com- as until adult (t ¼ 0.479, P ¼ 0.642, df ¼ 10, t-test) (Supp Fig. 1 [on- parison indicated that the model consisting of only the food- line only]). The mean developmental period was 27.6 6 0.58 d combination effect was selected as the best fit model in Experiments

(6 SD, N ¼ 6) in the Dm þ Pi1, 2, 5 treatment and 28.3 6 0.47 d 2 and 4 (Supp Table S1 [online only]), whereas the best fit model

(6 SD, N ¼ 6) in the Dm þ Pi1, 2, 3 treatment; there was no signifi- contained only the blood effect of water striders in Experiment 3 cant difference between the two treatments (t ¼2.08, P ¼ 0.0644, (Supp Table S1 [online only]). df ¼ 10, t-test). There was no significant difference in fertility between the two treatments (Fig. 2; t ¼ 2.01, P ¼ 0.0721, df ¼ 10, Discussion t-test).

In F2 generation, however, a substantial number of offspring The present results show that the combinations of food insects used failed to hatch and almost all F2 first instar nymphs did not reach to rear L. f. fossarum in the laboratory drastically alter viabilities in Journal of Insect Science, 2016, Vol. 16, No. 1 5 progeny generations. Although the origin of parental populations Zeller) to maintain predatory heteropteran insects for biological varied among the experiments, we compared the viability of off- control purposes (for a review, see De Clercq et al. 2014). This study spring using a split design in each experiment; thus, the differences further shows that P. interpunctella could also be a useful food insect in viabilities between treatments with different food insect for rearing water striders like crickets. combinations are ascribed to the effect of the food insect combin- The present results clearly indicate that adding nutrients to a ation. The developmental period is also influenced by food insects, Drosophila medium improves the viability of water striders. When suggesting the importance of the food insects in shortening the gen- L. f. fossarum is reared by our present method with D. melanogaster eration time. The present results further indicate that the combin- reared on the normal medium, one of the most frequent causes of ations of food insects in larval periods indeed affect wing morph death in nymphal stages is failed molting. Because cholesterol is the determination of adults in L. f. fossarum (Supp Table S1 [online precursor to ecdysone (an insect molting hormone) (Lang et al. only]) as reported in other water striders (Harada and Nishimoto 2012; Niwa and Niwa 2014), adding cholesterol to the medium for 2007). They strengthen the importance of establishing a continuous Drosophila could aid the synthesis of ecdysone in water striders, re- rearing method under constant diet conditions for future genetic sulting in successful molting. Although the viability until adulthood studies using L. f. fossarum. in the DmC þPi1, 2, 5 treatment in the Experiment 4 was about 20% For genetic studies such as linkage analysis and quantitative in the F2 generation, this low viability could be due to the use of la- traits loci (QTL) mapping, establishing segregating generations (e.g. boratory lines showing inbreeding depression. Indeed, results of F or backcross generations) is crucial. We have demonstrated that 2 Experiment 3 that used less inbred individuals recovered above the viability of F progenies depended on the F (i.e., parents of F 2 1 2 50% \viability until adulthood in the F generation even in the progenies) instars that fed on P. interpunctella adults, and the major- 2 Dm þ Pi treatment. Thus, the more than 50% viability in ity of F individuals from the Dm þ Pi treatment died before 1, 2, 5 2 1, 2, 3 Experiment 3 coupled with the consistently higher viabilities in the the second instar due to failure to successfully hatch or consume Dm þPi treatment than in the Dm þ Pi treatment in food insects in the first instar. In contrast, the fact that nearly 80% C 1, 2, 5 1, 2, 5 Experiment 4 suggest that adding cholesterol to Drosophila medium of nymphs successfully developed to the second stadium in the is one of the effective ways to obtain sufficient number of individ- Dm þ Pi1, 2, 5 treatment strongly indicates that not only the combination of food insects but also instars feeding on P. interpunctella in uals in segregating generations. Mayntz and Toft (2001) have also reported that the nutrient the F1 generation are very important in order to obtaining a sufficient number of individuals in the segregating generation in L. f. composition of rearing medium for a food insect (D. melanogaster) fossarum. can affect predator viability in the wolf spider Paradosa amentata Sonoda et al. (1991) reported that the viability of the broad (Clerck) (Araneae: Lycosidae). Therefore, adding nutrients to shouldered water strider, Microvelia douglasi Scott (Hemiptera: Drosophila medium could be a universal method for rearing preda- Veliidae), which is a polyphagous predator, is also affected by the cious insects. combination of food insects in the nymphal stage. Nymphs of M. This study does not intend to assess the genetic backgrounds of douglasi that fed on both D. melanogaster and Nilaparvata lugens wing polymorphisms in L. f. fossarum, but we preliminarily infer (Sta˚l) (which belong to different insect orders: Diptera and the contribution of genetic components (i.e., broad sense heritabil- 2 Hemiptera) develop faster and recover higher viability until adult- ity, H ) using the present rearing method (Dm þ Pi1, 2, 5). We calcu- hood than do nymphs that fed only on either D. melanogaster or N. lated the broad sense heritability of wing polymorphism as H2 ¼2r2 lugens (Sonoda et al. 1991). Several other studies have also demon- (among sibling groups)/[r2 (among sibling groups) þ r2 (within sib- strated that a mixed-prey diet in nymphal stages improved the adult ling groups)] (Ueno et al. 2001; Fukunaga and Akimoto 2007). We body size, fecundity, and the hatching success in offspring (Toft used the four inbred lines established by one-time sib-mating and we 1995; Grundy et al. 2000; Zanuncio et al. 2001; Marques et al. split the offspring from each sibling group fifty-fifty into two photo- 2015; for reviews, Lefcheck et al. 2013 and De Clercq et al. 2014), periods (16:8 (LD) h and 10:14 (LD) h). The mean square of vari- and such advantages of mixed diets have been already explained by ance obtained between sibling groups (163.4) is higher than that the toxin-dilution hypothesis (Freeland and Janzen 1974; Toft and within sibling groups (102.7), although we do not have enough sib- Wise 1999) and/or the redress-nutritional imbalance hypothesis ling group replications to test statistical significance. The estimated (Mayntz et al. 2005). Thus, the present results add to the growing broad sense heritability is 46%, and this indicates that a substantial number of cases showing the suitability of mixed-prey diets for amount of variation of wing polymorphism is determined by genetic predatory arthropods. factors in L. f. fossarum. Thus, further improvement of the present Crickets (e.g., Acheta domestica (L.) or Gryllus firmus Scudder) rearing method, especially for the nutrients to be included in the are often used as food insects for water striders in addition to dip- Drosophila medium, will contribute to elucidating the genetic mech- teran species (e.g., Fairbairn and King 2009). Our present rearing anisms determining wing polymorphisms and make L. f. fossarum a method using P. interpunctella with D. melanogaster recovered the model organism for studying the genetic background of wing same viability until adulthood when compared to the use of A. polymorphism. domestica with D. melanogaster in the same split design, although we conducted only one replicate in F1 generation (60% in both combinations, data not shown). P. interpunctella is readily reared in the Acknowledgments laboratory using brown rice as a food. We usually introduce two The authors thank A. Ejima and Y. Ishikawa for providing D. melanogaster adult pairs of P. interpunctella into a plastic container (135 85 strains, T. Kawamura for providing P. interpunctella, R. Masuda for his sup- 30 mm) filled with ca. 10 mm brown rice to generate offspring and port during the ﬁeldwork collecting L. f. fossarum, S. Nakao for habitat infor- generally obtain 50–120 adult offspring within 1 month without mation for L. f. fossarum, R. Niwa for information on the synthesis pathway any subsequent care after introduction (Hirooka pers. obs.). P. inter- of ecdysone, D. Hembry for English editing and comments on the draft manu- punctella is widely used as factitious prey with other pyralid moths script, and three anonymous reviewers for their invaluable comments on the (e.g., Corcyra cephalonica (Stainton) and Ephestia kuehniella manuscript. This study was supported by the Young Researcher Incubation 6 Journal of Insect Science, 2016, Vol. 16, No. 1

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