a colony was established from soybean and cowpea field collections of n. viridula in stoneville , ms , during the spring and the summer of 2010 . this colony has been maintained in culture for 5 years at the usda - ars national biological control laboratory . after approximately 40 generations in culture , three orange morph adult males were detected in the colony . the orange morph males were paired with green females at a two females per male sex ratio to represent a parent population ( f0 ) , and allowed to reproduce using the rearing methods described by rojas and morales - ramos ( 2014 ) . cages constructed from clear plastic boxes ( l 320  w 260  h 100  mm , part no . 048-c , pioneer packing , dixon , ky ) were modified with ten windows ( 27  mm dia . ) on the sides and four windows ( 65  mm dia . ) on the top all covered with nylon screen ( mesh 500 m ) . all adults in this study were reared in an environmentally controlled room at 26    1  c , 50    5% rh , and 14  h photophase and provisioned with peanuts , broccoli , a diet supplement , and fresh bean pods and a diet supplement that was replaced at 3 days intervals . eggs were collected daily and hatching nymphs ( f1 ) were allowed to develop under same conditions . the number of males and females of each coloration type was recorded for each generation in this study . resulting f1 adults were paired and reared at the same conditions to obtain the f2 generation . resulting f2 adults consisted of green females and green and orange males . only f2 orange males ( app . 25% of the total numbers ) were retained to mate with f2 females ( at two females per male sex ratio ) to produce the f3 . eggs were collected daily and enclosing nymphs were reared to the adult stage under the same conditions described above . because the f3 colony had become considerably larger , only resulting f3 adults from the first three emerging dates were counted and sexed . phenotype and sex frequencies observed in each generation were compared with frequencies expected , assuming that the orange color phenotype was determined by a single sex - linked recessive allele as reported by follett et al . the  test was used to compare observed versus expected frequencies within each generation cross . resulting f3 adults were divided to form two different colonies : orange type and green type . the pure orange - type colony was established using solely f3 orange - type adults . the f4 pure orange - type colony was used to generate a pure orange - type f5 . the reproductive potential of the f5 pure orange colony was compared with that of the pure green stock ( parental ) colony . newly emerging f5 orange - type adults were collected and grouped in cohorts of the same emerging date . a total of three cohorts were obtained from the green - type stock colony ( 209 adults total ) and four cohorts from the f5 orange type ( 168 adults total ) . adults were reared as described above and were monitored for oviposition and mortality , which were recorded daily . egg masses collected each day were recorded , labeled , and allowed to develop for 3 days before counting the eggs . embryos in developing eggs at 3 days old showed noticeable red eye coloration , which made them visible and allowed discrimination between fertilized and unfertilized eggs . the fertility ratio was calculated as fertile eggs / total eggs and fertility ratio was compared between color types using the z test . the mean number of eggs per mass was compared between treatments using anova and the student s t test . fertility tables were produced for each type using the methods reported by portilla et al . the f1 generation resulted in only green morphs ( 590  and 684  ) as expected from a recessive orange allele of single gene character . however , the f2 generation produced 672 green females , 351 green males , 298 orange males , and no orange females . these ratios were not significantly different to those expected for a recessive allele of a sex - linked gene ( table 1 ) and were consistent with follett et al . the first three cohorts of the f3 generation consisted of 345 green females , 346 green males , 100 orange females , and 85 orange males . the difference between observed and expected ratios were significant ( =8.067 , df  =  3 , p  =  0.046 ) ( table 1 ) , but this difference could be explained by significant deviations from the expected sex ratio of the f3 orange types ( |z|  =  2.38 , p  =  0.0087 ) resulting in a slight bias of 1.17 females per male . a similar female bias sex ratio was observed by follett et al . ( 2007 ) and this bias was explained as higher mortality in males occurring during their development . table 1.expected progeny ratios and observed frequencies in resulting f1 , f2 , and f3 generations after cross of orange males and green females in parent generation ( f0).expected phenotype ratios in progenyobserved phenotype frequencies in progenygeneration : parent and ( progeny)assumed genotypesg g o o g g o o f0 ( f1)xx x xy0.50.500590684006.936f1 ( f2)xx x xy0.50.2500.2567235102984.653f2 ( f3)0.5 xx x xy  +  0.5 xx x xy0.3750.3750.1250.125345346100858.067*degrees of freedom ( df )  =  3 ;  values with  *  show significant deviation from expected ratios.f2 green males were removed leaving only orange males to cross with green females . expected progeny ratios and observed frequencies in resulting f1 , f2 , and f3 generations after cross of orange males and green females in parent generation ( f0 ) . degrees of freedom ( df )  =  3 ;  values with  *  show significant deviation from expected ratios . mean oviposition rate (   sem ) measured as eggs per female per day was significantly higher in green - type stock colony females ( 2.76    0.29 ) as compared with f5 orange - type females ( 1.17    0.27 ) ( |t|  =  2.456 ; df  =  222 ; p  =  0.0148 ) . there was no significant difference in the number of eggs per egg mass between the parental stock green ( 27.14    1.26 ) and f5 orange ( 26.39    1.62 ) types . also , the proportion of fertile eggs was not significantly different between green ( 0.718    0.0094 ) and orange ( 0.726    0.0058 ) types . however , green - type females produced significantly higher number of egg masses per day ( 2.09    0.16 ) than orange type females ( 0.71    0.15 ) ( |t|  =  6.14 ; df  =  222 ; p  <  0.0001 ) . the reproductive output of green - type females was higher as measured by the ro value ( 20.67 ) compared with orange - type females ( 13.71 ) ( fig . in addition to the reduced expected phenotypic ratio in natural populations ( consisting mostly of males due to the sex linked and recessive nature of the orange allele ) , the lower reproductive output by orange types may be an explanation for their rarity in natural populations . 1.cumulative lxmx ( reproductive output ) from fertility tables of green and pure orange types of nezara viridula . cumulative lxmx ( reproductive output ) from fertility tables of green and pure orange types of nezara viridula .