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 .
 nezara viridula adult coloration can vary , including a rare orange - colored type ( i.e. , n. viridula f. aurantiaca ) . in november 2015 
 , three nezara viridula males displaying orange coloration were found in an established colony in stoneville , ms . 
 the objectives of this study were to determine if alleles of these orange types conformed to the allele characteristics previously reported for n. viridula f. aurantiaca and to determine if there were any differences in reproductive output compared with the green - colored type . 
 the three orange - type males were crossed with green - type females to produce a hybrid f1 . the f1 progeny was allowed us to cross to produce an f2 . 
 the f2 progeny consisted of 672 green females , 351 green males , 298 orange males , and 0 orange females . 
 these ratios did not differ significantly from the expected 50:25:25:0 ratios for a single recessive sex linked allele for color phenotype . 
 the f2 cross of green females and orange males produced an f3 consisting of 345 green females , 346 green males , 100 orange females , and 85 orange males . 
 these ratios also conformed to the expected ratios ( 0.375:0.375:0.125:0.125 ) with the exception of orange males , which numbers were slightly lower than expected . 
 the pure orange type n. viridula produced significantly less egg masses ( 0.71    0.15 ) per day than green types ( 2.09    0.16 ) and their reproductive output , measured as net reproductive rate ( ro ) , was lower in orange ( 13.71 ) compared with green ( 20.67 ) types . 
Materials and Methods
Results and Discussion