insects : adults of l. f. fossarum ( p generation ) were collected from ishigaki island ( 2422 - 27n , 1249 - 12e ) , okinawa prefecture , ryukyu archipelago , japan .
twenty - six females and 16 females were obtained on 3 april and 13 august 2012 , respectively .
collected adult females were maintained with d. melanogaster as food and allowed to lay eggs at 25  c under a 16:8 ( l : d ) h photoperiod in the laboratory of kyoto prefectural university , and obtained eggs were used for the subsequent experiments .
we also established a laboratory population from two females sampled in spring and 16 females sampled in summer 2012 , and used these for the experiments .
as food insects , two species of drosophila ( d. melanogaster and d. hydei sturtevant ) were maintained on a medium jazz - mix ( fisher scientific inc . ,
tokyo , japan ) at 25  c under a 16:8 ( ld ) h photoperiod in the laboratory .
we maintained two wild types ( oregon - r and canton - s ) and two mutants , curly ( cy ) and vestigial ( vg ) of d. melanogaster , and randomly used these as food for l. f. fossarum .
in addition to the two drosophila species , p. interpunctella were collected near the campus of kyoto prefectural university ( shimogamo , sakyo , kyoto , japan ) .
adult pairs of p. interpunctella were introduced to single clear plastic containers ( 135  85  30  mm ) containing brown rice as larval food and maintained at 28  c under a 16:8 ( ld ) h photoperiod in the laboratory . emerged adults of all these food insects were killed by freezer and used as prey in experiments of food insect combinations .
general 
 rearing 
 method : eggs from each collected wild female or the laboratory population were incubated on a water - soaked filter paper in plastic petri dishes ( 52  mm diameter , 13  mm depth ) .
newly hatched nymphs were introduced to translucent plastic containers ( 60  mm  60  mm  45 mm ) in groups of up to five individuals from the same parent .
these nymphs were then transferred individually to other clear plastic containers ( 60  mm  60  mm  95  mm ; plant box , # cul - jar300 , agc techno glass co. , ltd . ,
tokyo , japan ) at the third or fourth instar depending on the subsequent experimental design .
each container was filled with 5  mm water that had been dechlorinated by being incubated over 24  h in the laboratory .
a cut piece of extruded polystyrene plate was added on the water surface as a resting site .
all food insects , polystyrene plates , and water in the rearing containers were replaced daily regardless of what food insects were provided to the water striders .
eggs and nymphs were maintained at 25  c under a 16:8 ( ld ) h photoperiod in all experiments .
food 
 insect 
 combinations : to assess effective food insect combinations for rearing l. f. fossarum successively , we estimated viability from f1 eggs to adults , fertility of f1 adults ( i.e. germination rates of f2 eggs ) , hatchability of f2 nymphs , and viability of f2 nymphs until the second instar or adults with the following four food insect experiments .
detailed names of food insect species and the amount of each food insect used per day in each experiment are shown in table 1 . 

table 1 . species and amount of adult food insects fed to each stage of l. f. fossarumrearing methods1st2nd3rd4th5thadultexperiment 1dm+dhdm 1 dm 2 dm 3dh 1dh 2dh 3experiment 2dmdm 1 dm 2 dm 2 dm 3 dm 3 dm 4dm+pi1 , 2 dm 1 , pi 1/3 dm 2 , pi 1/2 dm 2 dm 3 dm 3 dm 4experiment 3dm+pi1 , 2 , 3 dm 1 , pi 1/3 dm 2 , pi 1/2 dm 2 , pi 1 dm 3 dm 3 dm 4dm+pi1 , 2 , 5 dm 1 , pi 1/3 dm 2 , pi 1/2 dm 2 dm 3 dm 3 , pi 1 dm 4experiment 4dm+pi1 , 2 , 5 dm 1 , pi 1/3 dm 2 , pi 1/2 dm 2 dm 3 dm 3 , pi 1 dm 4dmc+pi1 , 2 , 5dmc 1 , pi 1/3dmc 2 , pi 1/2dmc 2dmc
3dmc 3 , pi 1dmc 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 .  
species and amount of adult food insects fed to each stage of l. f. fossarum 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 .
f1 offspring derived from 26 females from the spring samples ( collected on 3 april 2012 ) were used in this experiment .
hatched f1 nymphs were fed on two species of drosophila ( dm  +  dh treatment , table 1 ) .
f1 viability was estimated as the percentage survival of individuals until adulthood out of all eggs obtained .
f1 offspring which successfully survived to adulthood were crossed with other adults from the same parent ( i.e. , full - sib mating ) and f2 eggs were obtained .
thus , we checked for the presence of eyespots on each egg 6 d after oviposition , and the presence of eyespots was regarded as an indicator of successful egg germination .
experiment 2 : p. interpunctella for 1st and 2nd instars in addition to d. melanogaster for all instars .
f1 offspring from 16 females from the summer samples ( collected on 13 august 2012 ) were used for this experiment . to assess the effect of adding p. interpunctella as a food insect , 20 eggs
each from 16 females were split 1:1 into two food treatments . in the first treatment ( dm treatment )
, l. f. fossarum nymphs were fed only on d. melanogaster for all stages ; in the second treatment ( dm  +  pi1 , 2 treatment ) , the nymphs were fed daily , alternating between d. melanogaster and p. interpunctella , until the end of the second instar and then fed only on d. melanogaster until adulthood ( table 1 ) . in each treatment , we estimated f1 viability as the mean percentage of individuals surviving until each instar or adulthood of 16 sibling groups and compared the viabilities of the two treatments in each stage .
we also compared f1 developmental periods from hatching to adult eclosion using f1 offspring that successfully became adults .
we crossed f1 offspring from four randomly selected sibling groups and obtained 50 f2 generation eggs from each group .
we counted fertile eggs of the f2 generation using the same method as in experiment 1 .
we assessed f2 hatchability and viability until second instar only in the dm  +  pi1 , 2 treatment 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 experiment ,
six females that had started oviposition ( p generation ) were isolated individually into plant boxes and were allowed to lay eggs to produce f1 offspring . to assess to which instars it would be most effective to provide p. interpunctella as food , 20 f1 eggs each from six females were split 1:1 into the dm  +  pi1 , 2 , 3 treatment and dm  +  pi1 , 2 , 5 treatment ( table 1 ) . in the f1 generation , we recorded viability until adulthood from eggs , length of developmental period from first instar to adult , and fertility of f1 adults . in the f2 generation , for each treatment , we recorded nymph hatchability and viability until the second instar in the same manner as in experiment 2 .
each parameter was compared between the dm  +  pi1 , 2 , 3 and dm  +  pi1 , 2 , 5 treatments .
we only assessed f2 viability until adulthood in the progeny of the dm  +  pi1 , 2 , 5 treatment due to the very low survivorship of nymphs from the dm  +  pi1 , 2 , 3 treatment ( see results ) .
experiment 4 : d. melanogaster reared on cholesterol - added medium for all instars and p. interpunctella for first , second , and fifth instars .
this experiment also used the laboratory population of l. f. fossarum and five females that had started oviposition ( p generation ) were isolated individually into plant boxes and were allowed to lay eggs to produce f1 offspring . to assess the effect of using d. melanogaster reared on a cholesterol - added medium ( table 2 ) as a food insect , 20 f1 eggs each from five females were split 1:1 into the dm  +  pi1 , 2 , 5 treatment and dmc  +  pi1 , 2 , 5 treatment ( table 1 ) .
we recorded the same parameters as in experiment 3 ( viability until adulthood from eggs , length of developmental period from first instar to adult , and fertility of adults in the f1 generation ; nymph hatchability , viability until the second instar , and viability until adulthood in the f2 generation ) , and each parameter was compared between the dm  +  pi1 , 2 , 5 and dmc  +  pi1 , 2 , 5 treatments . 

ingredients in the cholesterol - added medium for drosophila melanogaster in the experiment 4 ( amounts per 100 culture bottles)materialamountmedium ( jazz - mix)68  gcholesterol0.36  gdistilled water340  ml fisher scientific inc . , illinois , usa .
ingredients in the cholesterol - added medium for drosophila melanogaster in the experiment 4 ( amounts per 100 culture bottles )   fisher scientific inc . ,
osaka , japan . statistical analysis : we used student s t - test in all analyses for split - design experiments in experiments 24 .
however , we used welch s t - test for the hatchability and viability of the f2 generation in the experiment 4 due to the unequal variances between the dm  +  pi1 , 2 , 5 and dmc  +  pi1 , 2 , 5 treatments . in every analysis , we used arcsine square - root - transformed the viability , fertility and hatchability per sibling group in the f1 or f2 generation in order to satisfy the requirement of normality .
we also analyzed the effect of food - insect combinations on wing morphs ( macropterous , micropterous , and apterous ) by using the 
all statistical analyses were carried out using the r version 3.2.3 ( r developmental core team 2015 ) .
experiment 1 : d. melanogaster and d. hydei : of 658 f1 eggs , only 102 individuals survived until adulthood ( viability 15.5% ) .
although 1218 f2 eggs were laid by 14 f1 females , there were no fertile eggs under these food conditions .
thus , we could not assess the hatchability and viability of the f2 generation , suggesting that we could not rear l. f. fossarum continuously using only drosophila species as food insects .
experiment 2 : p. interpunctella for 1st and 2nd instars in addition to d. melanogaster for all instars : in the dm  +  pi1 , 2 treatment , over 70% of individuals successfully survived to adulthood ( fig .
on the other hand , viability decreased suddenly after the fourth instar mainly due to failure to molt , and in 10 of 16 sibling groups , no individuals survived until adulthood .
nymphal viability was significantly higher in the dm  +  pi1 , 2 treatment than in the dm treatment starting in the fourth instar ( fig . 1 ; until second instar t  =  0.206 , p  =  0.839 ; until third instar t  =  0.124 , p  =  0.902 ; until fourth instar t  =  2.33 , p  =  0.0270 ; until fifth instar t  =  6.25 , p  =  6.97e7 ; until adulthood t  =  11.8 , p  =  8.52e13 ; df  =  30 for all t - tests ) . 
 fig .
1 . comparison of survival rates between f1 generations fed on plodia in first and second instars ( dm  +  pi1 , 2 ) and fed only on drosophila in all instars and adulthood ( dm ) .
p  >  0.05 , * p  <  0.05 ( t - test )
. 
   comparison of survival rates between f1 generations fed on plodia in first and second instars ( dm  +  pi1 , 2 ) and fed only on drosophila in all instars and adulthood ( dm ) .
p  >  0.05 , * p  <  0.05 ( t - test ) .
the mean developmental period was 27.6    1.62 d (   sd , n  =  17 ) in the dm  +  pi1 , 2 treatment , whereas it was 29.6    2.43 d (   sd , n  =  7 ) in the dm treatment ; there was a significant difference between the two treatments ( t  =  2.35 , p  =  0.0279 , df  =  22 , t - test ) . in the dm  +  pi1 , 2 treatment , although the mean fertility of f1 adults was above 90% , in the subsequent f2 generation , mean hatchability and viability until the second instar decreased to 53.9    40.5% and 0.52    1.04% , respectively (   sd ) . in the dm treatment ,
no fertile eggs were obtained as in experiment 1 , although a single f1 female started oviposition .
experiment 3 : p. interpunctella for third or fifth instars in addition to the dietary conditions from experiment 2 .
f1 viabilities until adulthood were high ( dm  +  pi1 , 2 , 3  =  80.0% , dm  +  pi1 , 2 , 5  =  83.3% ) in both treatments , and there were no significant differences between the two treatments in viabilities until each instar ( until second instar t  =  0.202 , p  =  0.844 ; until third instar t  =  0.483 , p  =  0.639 ; until fourth instar t  =  0.661 , p  =  0.524 ; until fifth instar t  =  0.788 , p  =  0.449 ; df  =  10 for all tests ; t - test ) as well as until adult ( t  =  0.479 , p  =  0.642 , df  =  10 , t - test ) ( supp fig . 1 [ online only ] ) .
the mean developmental period was 27.6    0.58 d (   sd ,
n  =  6 ) in the dm  +  pi1 , 2 , 5 treatment and 28.3    0.47 d (   sd , n  =  6 ) in the dm  +  pi1 , 2 , 3 treatment ; there was no significant difference between the two treatments ( t  =  2.08 , p  =  0.0644 , df  =  10 , t - test ) .
2 ; t  =  2.01 , p  =  0.0721 , df  =  10 , t - test ) . 

comparison of the fertility and survival rate between f2 generations produced by f1 offspring fed on plodia in the first through third instars ( dm  +  pi1 , 2 , 3 ) and in the first , second and fifth instars ( dm  +  pi1 , 2 , 5 ) .
p  >  0.05 , * p  <  0.05 ( t - test ) . 
  
comparison of the fertility and survival rate between f2 generations produced by f1 offspring fed on plodia in the first through third instars ( dm  +  pi1 , 2 , 3 ) and in the first , second and fifth instars ( dm  +  pi1 , 2 , 5 ) .
p  >  0.05 , * p  <  0.05 ( t - test ) . in f2 generation , however , a substantial number of offspring failed to hatch and almost all f2 first instar nymphs did not reach the second stadium in the dm  +  pi1 , 2 , 3 treatment ( fig .
in the dm  +  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 viability between the two treatments ( hatchability t  =  3.42 , p  =  0.00653 , viability until second instar t  =  8.89 , p  =  4.62e6 , df  =  10 , t - test ) . the mean percentile viability until adulthood in the dm  +  pi1 , 2 ,
5 treatment was 53.0    23.6 (   sd , n  =  4 ) .
experiment 4 : d. melanogaster reared on cholesterol - added medium for all instars and p. interpunctella for first , second , and fifth instars : f1 viabilities until adulthood were not significantly differentiated between the two treatments ( dm  +  pi1 , 2 , 5  =  70.0% , dmc  +  pi1 , 2 , 5  =  74.0% ; t  =  0.410 , p  =  0.693 , df  =  8 , t - test ) as in experiment 3 ( supp fig .
the mean developmental period was 28.6    0.49 d (   sd ,
n  =  5 ) in the dm  +  pi1 , 2 , 5 treatment and 28.3    0.74 d (   sd , n  =  5 ) in the dmc  +  pi1 , 2 , 3 treatment ; there was no significant difference between the two treatments ( t  =  0.65 , p  =  0.533 , df  =  8 , t - test ) .
3 ; t  =  0.7244 , p  =  0.490 , df  =  8 , t - test ) . 

comparison of the fertility and survival rate between f2 generations fed on normal d. melanogaster ( dm  +  pi1 , 2 , 5 ) and on d. melanogaster reared on the cholesterol - added medium ( dmc  +  pi1 , 2 , 5 ) .
p  >  0.05 , * p  <  0.05 ( welch 's t - test ) . 
  
comparison of the fertility and survival rate between f2 generations fed on normal d. melanogaster ( dm  +  pi1 , 2 , 5 ) and on d. melanogaster reared on the cholesterol - added medium ( dmc  +  pi1 , 2 , 5 ) .
p  >  0.05 , * p  <  0.05 ( welch 's t - test ) . in f2 generation ,
hatchability and viability until each instar and adulthood are consistently higher in the dmc  +  pi1 , 2 , 5 treatment than in the dm  +  pi1 , 2 , 5 treatment , although significant differences between the two treatments are detected in the viability until adulthood alone ( fig .
3 ; hatchability t  =  1.003 , p  =  0.346 , df  =  7.825 , viability until second instar t  =  1.727 , p  =  0.146 , df  =  4.891 ; until third instar t  =  1.357 , p  =  0.243 , df  =  4.185 ; until forth instar t  =  1.386 , p  =  0.233 , df  =  4.352 ; fifth instar t  =  2.353 , p  =  0.0515 , df  =  6.877 ; until adulthood t  =  2.484 , p  =  0.0381 , df  =  7.935 ; welch 's t - test ) . there was no significant difference in the mean larval developmental period between the dmc  +  pi1 , 2 , 5 treatment ( 42.4    2.54 d [   sd , n  =  5 ] ) and the dm  +  pi1 , 2 , 5 treatment ( 44.2    2.36 d [   sd , n  =  3 ] ) ( t  =  1.00 , p  =  0.355 , df  =  6 , t - test ) . effect of food - insect combinations on wing morphs : model comparison indicated that the model consisting of only the food - combination effect was selected as the best fit model in experiments 2 and 4 ( supp table s1 [ online only ] ) , whereas the best fit model contained only the blood effect of water striders in experiment 3 ( supp table s1 [ online only ] ) .
the present results show that the combinations of food insects used to rear l. f. fossarum in the laboratory drastically alter viabilities in progeny generations .
although the origin of parental populations varied among the experiments , we compared the viability of offspring using a split design in each experiment ; thus , the differences in viabilities between treatments with different food insect combinations are ascribed to the effect of the food insect combination .
the developmental period is also influenced by food insects , suggesting the importance of the food insects in shortening the generation time .
the present results further indicate that the combinations of food insects in larval periods indeed affect wing morph determination of adults in l. f. fossarum ( supp table s1 [ online only ] ) as reported in other water striders ( harada and nishimoto 2007 ) .
they strengthen the importance of establishing a continuous rearing method under constant diet conditions for future genetic studies using l. f. fossarum . for genetic studies such as linkage analysis and quantitative traits loci ( qtl ) mapping , establishing segregating generations ( e.g. f2 or backcross generations )
we have demonstrated that the viability of f2 progenies depended on the f1 ( i.e. , parents of f2 progenies ) instars that fed on p. interpunctella adults , and the majority of f2 individuals from the dm  +  pi1 , 2 , 3 treatment died before the second instar due to failure to successfully hatch or consume food insects in the first instar . in contrast , the fact that nearly 80% of nymphs successfully developed to the second stadium in the dm  +  pi1 , 2 , 5 treatment strongly indicates that not only the combination of food insects but also instars feeding on p. interpunctella in the f1 generation are very important in order to obtaining a sufficient number of individuals in the segregating generation in l. f. fossarum .
( 1991 ) reported that the viability of the broad shouldered water strider , microvelia douglasi scott ( hemiptera : veliidae ) , which is a polyphagous predator , is also affected by the combination of food insects in the nymphal stage .
nymphs of m. douglasi that fed on both d. melanogaster and nilaparvata lugens ( stl ) ( which belong to different insect orders : diptera and hemiptera ) develop faster and recover higher viability until adulthood than do nymphs that fed only on either d. melanogaster or n. lugens ( sonoda et al .
several other studies have also demonstrated that a mixed - prey diet in nymphal stages improved the adult body size , fecundity , and the hatching success in offspring ( toft 1995 ; grundy et al .
2014 ) , and such advantages of mixed diets have been already explained by the toxin - dilution hypothesis ( freeland and janzen 1974 ; toft and wise 1999 ) and/or the redress - nutritional imbalance hypothesis ( mayntz et al .
thus , the present results add to the growing number of cases showing the suitability of mixed - prey diets for predatory arthropods . crickets ( e.g. , acheta domestica ( l. ) or gryllus firmus scudder ) are often used as food insects for water striders in addition to dipteran species ( e.g. , fairbairn and king 2009 ) .
our present rearing method using p. interpunctella with d. melanogaster recovered the same viability until adulthood when compared to the use of a. 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 ) .
we usually introduce two adult pairs of p. interpunctella into a plastic container ( 135  85  30  mm ) filled with ca .
10  mm brown rice to generate offspring and generally obtain 50120 adult offspring within 1 month without any subsequent care after introduction ( hirooka pers .
p. interpunctella is widely used as factitious prey with other pyralid moths ( e.g. , corcyra cephalonica ( stainton ) and ephestia kuehniella zeller ) to maintain predatory heteropteran insects for biological control purposes ( for a review , see de clercq et al .
this study further shows that p. interpunctella could also be a useful food insect for rearing water striders like crickets .
the present results clearly indicate that adding nutrients to a drosophila medium improves the viability of water striders .
when l. f. fossarum is reared by our present method with d. melanogaster reared on the normal medium , one of the most frequent causes of death in nymphal stages is failed molting . because cholesterol is the precursor to ecdysone ( an insect molting hormone ) ( lang et al .
2012 ; niwa and niwa 2014 ) , adding cholesterol to the medium for drosophila could aid the synthesis of ecdysone in water striders , resulting in successful molting .
although the viability until adulthood in the dmc  +  pi1 , 2 , 5 treatment in the experiment 4 was about 20% in the f2 generation , this low viability could be due to the use of laboratory lines showing inbreeding depression .
indeed , results of experiment 3 that used less inbred individuals recovered above 50% \viability until adulthood in the f2 generation even in the dm  +  pi1 , 2 , 5 treatment .
thus , the more than 50% viability in experiment 3 coupled with the consistently higher viabilities in the dmc  +  pi1 , 2 , 5 treatment than in the dm  +  pi1 , 2 , 5 treatment in experiment 4 suggest that adding cholesterol to drosophila medium is one of the effective ways to obtain sufficient number of individuals in segregating generations .
mayntz and toft ( 2001 ) have also reported that the nutrient composition of rearing medium for a food insect ( d. melanogaster ) can affect predator viability in the wolf spider paradosa amentata ( clerck ) ( araneae : lycosidae ) .
therefore , adding nutrients to drosophila medium could be a universal method for rearing predacious insects .
this study does not intend to assess the genetic backgrounds of wing polymorphisms in l. f. fossarum , but we preliminarily infer the contribution of genetic components ( i.e. , broad sense heritability , h ) using the present rearing method ( dm  +  pi1 , 2 , 5 ) .
we calculated the broad sense heritability of wing polymorphism as h=2 ( among sibling groups)/[ ( among sibling groups )  +   ( within sibling groups ) ] ( ueno et al .
we used the four inbred lines established by one - time sib - mating and we split the offspring from each sibling group fifty - fifty into two photoperiods ( 16:8 ( ld ) h and 10:14 ( ld ) h ) .
the mean square of variance obtained between sibling groups ( 163.4 ) is higher than that within sibling groups ( 102.7 ) , although we do not have enough sibling group replications to test statistical significance .
the estimated broad sense heritability is 46% , and this indicates that a substantial amount of variation of wing polymorphism is determined by genetic factors in l. f. fossarum .
thus , further improvement of the present rearing method , especially for the nutrients to be included in the drosophila medium , will contribute to elucidating the genetic mechanisms determining wing polymorphisms and make l. f. fossarum a model organism for studying the genetic background of wing polymorphism .
 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 ( hbner ) , 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 contaminations 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 . 
Materials and Methods
Results
Discussion
Supplementary Data