Genetics of a sex-linked recessive red eye color mutant of the tarnished plant bug, Lygus lineolaris

doi:10.4236/ojas.2013.32A001

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Vol.3, No.2A, 1-9 (2013) Open Journal of Animal Sciences

doi:10.4236/ojas.2013.32A001

Genetics of a sex-linked recessive red eye color

mutant of the tarnished plant bug, Lygus lineolaris

Margaret Louise Allen

Biological Control of Pests Research Unit, USDA Agricultural Research Service, Stoneville, USA;

meg.allen@ars.usda.gov

Received 5 April 2013; revised 9 May 2013; accepted 23 May 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

An inbred colony of the tarnished plant bug,

Lygus lineolaris (Palisot de Beauvois) (Miridae:

Hemiptera), was observed to contain specimens

with abnormal traits including red eyes, deform-

ed antennae, and deformed legs. These speci-

mens were isolated and back crossed to create

stable phenotypic strains. The only successful

strain established was a red eyed strain named

Cardinal. The trait was more prevalent and sta-

ble in males, suggesting that it could be sex

linked. To test the hypothesis that the trait was

based on a recessive sex linked allele, classical

genetic crosses were performed. The hypothe-

sis was confirmed, and the eye color pheno-

type was measured and characterized using

color analysis software. The trait is similar to

other red eyed phenotypes described in this

species, but is clearly based on a different mu-

tation since it is sex linked rather than auto-

somal. The results of crossing experiments also

suggest that inbreeding in this species result s in

substantial fitness cost to laboratory insects.

Keyw ords: Genetics; Inbreeding; Fitness;

Pigmentation; Ommochromes; Antennae; Spineless

1. INTRODUCTION

Variations in the eye color of insects can be found in

nature and selected by inbreeding or induced by muta-

genesis. Naturally occurring and induced mutations have

served as a foundation for progress in both general ge-

netics and in genetic manipulation of insects. A white eye

color mutation was found to be linked to sex in Droso-

phila melanogaster, defining the phenomenon of sex-

linkage, and establishing methods in classical genetics

[1]. Transplantation studies using D. melanogaster eye

disks from multiple eye color strains were vital to the

establishment of biochemical pathway genetics [2]. In-

sect eye colors were later found to be more complex than

could be explained by the one-gene-one-enzyme princi-

ple, and were shown to be related to transporter path-

ways [3]. More recently, eye color mutations have been

utilized extensively to verify manipulation systems in-

volving genetic transformation [4,5], especially when the

eye-specific promoter 3xP3 is utilized [6].

Eye color mutations have been described in numerous

insects other than D. melanogaster. Within the order

Diptera, eye color mutations have been described in fruit

flies, family Tephritidae [7-9], in blow flies, family Cal-

liphoridae [10-14] and house flies [15-17], in the tsetse

fly [18-22], and in many mosquitoes [23-33]. Eye color

mutations have also been described in the order Lepi-

doptera [34-40], in the order Coleoptera [41-43], and in

the order Hymenoptera [44-46]. In the order Hemiptera,

eye color mutations have been described in kissing bugs

(family Reduviidae) [47-51], in the tarnished plant bug,

the subject of this paper, family Miridae [52,53], and

recently in the brown planthopper, family Delphacidae

[54].

The rapid pace of progress in genomics and bioinfor-

matics presents remarkable opportunities for discovery

and analysis of insect genes. Efforts to capitalize on this

progress will be enhanced by increased focus on basic

biological function phenomena in insects that have not

yet been developed as model organisms. Naturally oc-

curring strains with genetically defined visible markers

will once again serve as valuable resources to scientific

progress. A red-eyed strain, established through inbreed-

ing of Lygus lineolaris, was reported previously [53]. A

similar strain, established from a wild male specimen

collected in the field in Arkansas was reported more re-

cently [52]. Through genetic analysis, the eye color allele

in these naturally occurring strains were characterized as

autosomal recessive.

M. L. Allen / Open Journal of Animal Sc iences 3 (2013) 1-9

As part of an effort to perform in vivo genetic ma-

nipulations on L. lineolaris, a laboratory strain was es-

tablished in quarantine and inbred for 5+ years. This

strain was examined regularly for unusual phenotypes

that might indicate mutation. Phenotypes associated with

abnormal appendages were observed but could not be

reproduced and maintained in the laboratory through

normal breeding. However, an eye color mutation was

observed and isolated. To verify that this mutant strain

was not the same as the ones previously described, re-

ciprocal crosses were undertaken to test the hypothesis

that the mutation was sex linked. Additionally, digital

image analysis was utilized to characterize the color of

the phenotype. To differentiate the phenotype from those

previously described, the strain was called Cardinal, with

the eye color mutation card inal. Strains generated by

inbreeding could be highly useful in forthcoming ge-

nomics projects using this species of pest insect and

other arthropods.

2. MATERIALS AND METHODS

2.1. Insects

A wild-type colony was maintained without introgres-

sion for 5+ years in the Stoneville Research Quarantine

Facility (SRQF) in Mississippi. Insect stocks were kept

in an environmental chamber (Percival Scientific, Inc.

Perry, IA) set for 16:8 (L:D) h lighting regime with 25˚C

daytime and 19˚C nighttime temperature and 55%RH.

Insects were provided a standardized diet and oviposition

system based on NI diet and 4% gelcarin oviposition

substrate [55].

2.2. Classical Genetics

For first generation matings small groups of virgin in-

sects, two males and four females, were housed in isola-

tion. Second generation insects were grouped in sets of

two males and seven females. Insects were sexed in the

5th instar [52]. Eggs were collected daily. Mature eggs

were separated from oviposition substrate and kept on

moist filter paper for observation until hatch, then trans-

ferred to 100 mm by 15 mm Petri dishes for rearing to

the adult stage. Nymphs were provisioned with fresh red

clover leaves as dietary supplement and refugia.

2.3. Imaging

Images of living specimens were collected using a

stereoscopic zoom microscope (Nikon SMZ1500) and

Nikon digital camera (DMX 1200). Images used to ana-

lyze the eye color were cropped to include only eyes and

converted to .jpg files then analyzed using RGB software

[56]. Each image was analyzed three times using differ-

ent portions of the eye image for analysis. The means of

these samples were then used to generate color match

indicators for illustrations.

2.4. Statistical Analyses

Egg production data were analyzed using the mixed

procedure SAS Enterprise Guide v. 4.2 (SAS 2006). Phe-

notype and sex ratio data were analyzed for goodness of

fit to Mendelian ratios using the Chi-Square test [57].

3. RESULTS

After inbreeding a culture of L. lineolaris without in-

trogression for overlapping generations of roughly 60

days for four years (approximately 24 generations), a red

eyed individual was identified in a colony cage. For the

next two years backcrossing and inspection of the parent

colony for additional red eyed stock eventually produced

a homozygous strain of red eyed specimens (Figure 1).

When the first red-eyed individuals were identified they

were invariably male, and were paired with wild type

females from the inbred source colony. When back

crosses of the offspring were obtained, male red eyed

individuals were found but red-eyed females were rare.

Thus the possibility that the eye color was a sex linked

phenotype was recognized early on. It took several gen-

erations to establish a homozygous colony of red-eyed

specimens (Cardinal), and the overall fitness of the strain

seemed low, compared to the wild type (empirical ob-

servation). As Cardinal strain was becoming established,

additional rare phenotypes began to appear regularly in

both the wild and Cardinal strains. These phenotypes

appeared as shortened and deformed appendages, ini-

tially short antennae (Figure 2). These individuals were

also backcrossed and crossed with one another, but the

phenotype was never reliably reproduced in offspring.

The most common deformity other than short antennae

found in the inbred colonies was truncation of legs (Fig-

ure 3). These deformities sometimes coincided with the

cardinal mutation, but also appeared in wild types with

no apparent linkage. Because the phenotypes could not

be established in permanent culture, no genetic data

could be collected.

To test the hypothesis that fecundity was lower in the

Cardinal strain, eggs were collected from small pools of

individuals of wild type, Cardinal, and reciprocal crosses

of the two strains. Egg production is shown in Ta bl e 1 .

While the differences in egg production were not sig-

nificant in the first five or ten days of the test, by day 15,

and continuing on day 20, the cumulative egg production

data met the threshold (P < 0.05) to signify statistically

significant difference (Table 1). Differences of least

squares means on day 20 showed no significance be-

tween homozygous Cardinal (Ca × Ca) egg production

and homozygous wild type (W × W) egg production (P =

0.262, t = −1.24, df = 6), and also no significant differ-

M. L. Allen et al. / Open Journal of Animal Sciences 3 (20 13 ) 1-9 3

Figure 1. Eye colors of inbred strains of Lygus lineolaris

(Palisot de Beauvois) (Hemiptera: Miridae) with square color

match indicator boxes. (A) Eggs produced by homozygous

insects: normal eye color above, cardinal eye color below; (B)

Eggs produced by crossing phenotypes: upper, normal eye

color; middle, cardinal eye color from crimson female parent;

lower, heterozygous (normal) eye color from crimson female

parent; (C) Young adult eye color comparison: left, cardinal;

right, normal (wild type); (D) Eye color of an older male (30+

days post adult eclosion/ecdysis).

ence between Ca x Ca and wild type male crossed with

Cardinal female (W × Ca) (P = 0.291, t = 1.16, df = 6)

nor between the W × W and W × Ca (P = 0.054, t =

−2.40, df = 6). Additionally, the egg production of recip-

rocal crosses (W × Ca/Ca × W) did not differ from one

another (P = 0.099, t = −1.95, df = 6). However, the Ca x

W cross produced significantly lower quantities of eggs

when compared to homozygous crosses W × W (P =

0.0048, t = −4.35, df = 6) and Ca × Ca (P = 0.021, t =

3.11, df = 6).

To test the hypothesis that the phenotype was sex

linked, crosses of the Cardinal and wild type strains were

evaluated, and yielded the expected ratios of offspring in

the first generation for a recessive sex-linked mutation

(Table 2A). All offspring of a male Ca parent were wild

type, while all male offspring from a female Ca parent

were red eyed and all female offspring were wild type.

Second generation crosses of heterozygous females from

both first generation crosses also yielded expected ratios

Figure 2. Inbred specimens of Lygus lineolaris that display

deformed antennae. (A)-(F) nymphs; (G) adult. Note that the

right middle leg of this specimen is also defective, tibia is

shortened and tarsal segments are fused.

of offspring (Ta bl e 2 B). The crosses are illustrated dia-

grammatically in Figure 4.

Overall survival to the imaginal stage from fertile em

bryos was only evaluated in the F1 mating pools: from

the male Ca parent 55.6% of wild type offspring survived

M. L. Allen / Open Journal of Animal Sc iences 3 (2013) 1-9

Table 1. Mean cumulative numbers of eggs (±SE) produced per female in crosses of inbred wild-type (W) and inbred cardinal-eyed

(Ca) specimens of Lygus lineolaris.

Cross Day

♂ × ♀ 5 na 10 n 15 n 20 n

W × W 13.08 ± 2.39 12 25.58 ± 2.1012 39.25 ± 2.2912 51.14 ± 4.29 10

Ca × Ca 12.33 ± 2.51 12 20.42 ± 2.9212 31.08 ± 3.2912 41.25 ± 3.44 12

W × Ca 7.33 ± 4.51 12 18.42 ± 8.4212 24.75 ± 10.0012 32.00 ± 11.40 11

Ca × W 5.33 ± 1.92 12 8.00 ± 2.74 12 12.58 ± 2.4312 16.42 ± 1.06 11

F 3.17 4.23 7.07 6.83

P > F 0.11 0.06 0.02 0.02

aThe degrees of freedom for each comparison was 3.39.

Table 2. First and second generation crosses of Cardinal eye color strain to parental wild type strain. (A) First generation heterozy-

gous female parents are either combined or identified by the sex of the Cardinal-eyed parent. Second generation combined analysis

included with pairwise comparisons of phenotype and male/female ratio. Critical value for all chi-square tests is 3.84, df = 1; (B)

Second generation crosses, combined data (above) and data analyzed by parent carrying cardinal allele. Critical value for phenotype

data is 7.82, df = 3.

A. adult phenotype adult survival

Cross egg phenotype χ2 wild type χ2 cardinal χ2 Totals χ2 Totals χ2

♂ × ♀ wild type cardinal ♂ ♀ ♂ ♀ wild typecardinal ♂ ♀

W × W 580 0 - 202 166 0.880 0 0 - 368 0 - 202 1660.880

Ca × Ca 0 537 - 0 0 - 248231 0.1510 479 - 248 2310.151

W × Ca 265 228 0.694 0 143 - 1360 - 143 136 0.044 136 1430.044

Ca × W 337 0 - 117 141 0.558 0 0 - 258 0 - 117 1410.558

Ca × F1 438 397 2.013 103 134 4.055a131125 0.141237 256 0.732 234 2591.268

B. adult phenotype adult survival

Cross egg phenotype χ2 wild type cardinal χ2 Totals χ2 Totals χ2

♂ × ♀ wild type cardinal ♂ ♀ ♂ ♀(df = 3) wild typecardinal ♂ ♀

Ca × F1 438 397 2.013 103 134 131 1254.777 237 256 0.732 234 2591.268

Ca × F1♂ 245 208 3.022 59 78 75 584.874 137 133 0.059 134 1360.015

Ca × F1♀ 193 189 0.041 44 56 56 674.749 100 123 2.372 100 1232.372

aExceeds critical value for chi-square tests, 3.84, df = 1.

and 64.9% of cardinal offspring survived; from the fe-

male Ca parent 69.5% of wild type offspring survived

and 70.5% of cardinal offspring survived. No significant

difference was detected (P > 0.05, student t-test, df = 2).

Eye colors of the wild-type embryonic insects corre-

sponded to RGB colors at R 193 ± 8, G 88 ± 7, B 70 ± 10

while the mutant embryonic eye colors corresponded to

RGB colors R 240 ± 2, G 130 ± 7, B 68 ± 10. These col-

ors persisted as developing embryos, nymphs, and

through early adult stage. As the adults darken with age,

particularly the males, the eyes darken to a more crimson

color corresponding to R 161 ± 7, G 70 ± 18, B 44 ± 20.

The fully mature wild-type adult eye corresponds to R

110 ± 7, G 58 ± 8, B 48 ± 6 (Figure 1).

4. DISCUSSION

Standardized laboratory rearing of L. lineolaris is pri-

marily for the purpose of bioassays. The cultures of in-

sects are expected to represent field performance of

naturally occurring populations. Thus, colonies are fre-

quently intermixed with new field collected insects in

order to maintain colony health and heterozygosity [58].

The laboratory colony in the SRQF differed in that no

M. L. Allen et al. / Open Journal of Animal Sciences 3 (20 13 ) 1-9 5

Figure 3. Individual specimen from Cardinal strain with severe

leg deformities. Four of six legs have shortened and deformed

termini. Tarsal segments appear to be fused. (A) Right lateral

view; (B) Ventral view; (C) Left middle leg terminus; (D) Right

hind leg; ( E) Right foreleg and middle leg termini.

introgression by field collected insects was allowed. This

culture strategy was specifically intended to limit het-

erozygosity and provide more inbreeding to support ge-

netic analysis and genetic manipulation. Under labora-

tory inbreeding conditions, spontaneous mutations often

are revealed. Novel eye color phenotypes are not un-

common in colonies of insects. Those arising in colonies

of D. melanogaster kept by Thomas Morgan and his stu-

dents formed a foundation for modern genetics.

The truncated appendages observed repeatedly in the

inbred colony bear resemblance to defects described in D.

melanogaster and Tribolium castaneum related to the

transcription factor spineless [59-61]. Further careful

breeding of similar specimens will be required to deter-

mine whether these phenotypes are related. Unfortu-

nately, the specimens we found were delicate and gener-

ally infertile, and were discarded after multiple attempts

to establish a stable strain failed.

Naturally occurring orange or red eye color pheno-

types of Hemiptera have been described [47-50,52-54].

Eye pigments in two species of Hemiptera in the family

Reduviidae differ from one another, those from Triatoma

infestans Klug being composed primarily of xanthom-

matin [47], while the eye pigments of Rhodnius prolixus

(a)

(b)

(c)

Figure 4. Diagrams showing expected results from crossing

wild type insects with insects carrying an autosomal recessive

red eye trait (R) compared with results from crossing wild type

insects (WT) with insects carrying a sex linked recessive red

eye trait such as cardinal (ca). MP = allele from male parent, FP

= allele is from the female parent.

are composed primarily of ommins [62]. The only en-

zyme identified from L. lineolaris from the ommochrome

biosynthetic pathway is tryptophan oxygenase (TO)

(http://www.ncbi.nlm.nih.g ov/nuccor e/307634529 and

M. L. Allen / Open Journal of Animal Sc iences 3 (2013) 1-9

http://www.ncbi.nlm.nih.gov/protein/307634530, submit-

ted 10 August 2010, accessed 31 July 2012). This en-

zyme catalyzes the initial reaction in the conversion of

tryptophan to ommochromes, the pigments in insect eyes.

The white gene used to define sex linkage by Morgan

(1910) was shown to be a membrane pigment transporter

gene rather than a pigment synthesis enzyme gene (Sul-

livan and Sullivan 1975), and this may also be the case in

L. lineolaris. The genetic identity of TO has been char-

acterized in D. melanogaster [63], and the red flour bee-

tle Tribolium castaneum [64], and putative homologues

with a high similarity at the nucleotide level [65] can be

identified in the human body louse Pediculus humanus

corporis (XM_002423485.1, accessed from NCBI 16

July 2012) and the mountain pine beetle Dendroctonus

ponderosae (BT128347.1, accessed from NCBI 16 July

2012) [66]. While TO may be mutated in either the

autosomal or sex-linked phenotypes of red eyed L. lineo-

laris, any number of other biosynthesis or transport

genes may also be responsible.

Both naturally occurring eye color strains of L. lineo-

laris previously identified were characterized as auto-

somal recessive alleles. The wild collected eye color

variant [52] was reported to have little effect on behavior

or physiology, although because of the low field preva-

lence of the phenotype it was implied that there could be

a fitness disadvantage, possibly associated with mating

disadvantage based on decreased visual ability. The

red-eyed “R” strain was kept in culture for >5 years, but

has since been discarded (G. Snodgrass, personal com-

munication). The eye color mutation identified through

inbreeding [53] was not tested for physiological charac-

teristics, but was observed to display no obvious behav-

ioral or developmental differences from wild type. The

strain reported here and the progenitor wild type strain

both exhibited apparent loss of fitness. The 20 day cu-

mulative egg production reported for the wild caught red

strain was over 120 eggs/female, while the specimens

reported here produced less than half that in every cross

(Table 1). The loss of fecundity is most distinct in the

fecundity of the Ca × W cross. The reason for this loss of

fecundity is not known. Interestingly, the total number of

male vs. female wild type offspring produced by the Ca

× F1 crosses exceeded the critical value for the expected

m/f ratio (Ta ble 2A), while the overall phenotype distri-

bution fit the expected results (Table 2B). Inbreeding

depression is an accepted phenomenon, relevant to evo-

lution, conservation, and agriculture. Intentional in-

breeding is also expected to play a vital and dominant

role in future molecular genetics and functional genomic

research [67].

If genetic manipulation technology improves to a point

at which sex-specific applications such as the sterile in-

sect technique (SIT) can be applied to insects in the order

Hemiptera, identification of sex-specific genes and gene

regulation will become critical to project success. Visible

markers that can be induced by inbreeding or other meth-

ods of mutagenesis will facilitate progress towards these

applications, and towards a better understanding of the

genetics of these important pest insects.

5. ACKNOWLEDGEMENTS

The author extends appreciation to the laboratory technicians who

provided assistance and support in completing this work, Fannie M.

Byrd, and especially to Catherine L. Smith who performed a vital role

in setting up experiments and collecting data. Thanks go to Dr. Xixuan

Jin, Dr. William B. Walker III, and Dr. William Rodney Cooper for

reviewing a previous version of this manuscript and providing helpful

suggestions. The United States Government has the right to retain a

non-exclusive, royalty-free license in and to any copyright of this arti-

cle. This article reports the results of research only. Mention of a com-

mercial or proprietary product does not constitute an endorsement of

the product by the United States Department of Agriculture. USDA is

an equal opportunity provider and employer.

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