RNA interference‐mediated knockdown of the …...the Acyrthosiphon pisum genome suggested the...

RNA interference-mediated knockdown of theHalloween gene Spookiest (CYP307B1) impedes adulteclosion in the western tarnished plant bug, Lygushesperus

E. Van Ekert*, M. Wang†, Y.-G. Miao†, C. S. Brent* and

J. J. Hull*

*USDA-ARS Arid Land Agricultural Research Center,Maricopa, AZ, USA; and †Zhejiang University, Hangzhou,China

Abstract

Ecdysteroids play a critical role in coordinating insect

growth, development and reproduction. A suite of

cytochrome P450 monooxygenases coded by what

are collectively termed Halloween genes mediate

ecdysteroid biosynthesis. In this study, we describe

cloning and RNA interference (RNAi)-mediated knock-

down of the CYP307B1 Halloween gene (Spookiest) in

the western tarnished plant bug, Lygus hesperus.

Transcripts for Ly. hesperus Spookiest (LhSpot) were

amplified from all life stages and correlated well with

timing of the pre-moult ecdysteroid pulse. In adults,

LhSpot was amplified from heads of both genders as

well as female reproductive tissues. Heterologous

expression of a LhSpot fluorescent chimera in cul-

tured insect cells co-localized with a fluorescent

marker of the endoplasmic reticulum/secretory path-

way. RNAi-mediated knockdown of LhSpot in fifth

instars reduced expression of ecdysone-responsive

genes E74 and E75, and prevented adult development.

This developmental defect was rescued following

application of exogenous 20-hydroxyecdysone but

not exogenous 7-dehydrocholesterol. The unequivo-

cal RNAi effects on Ly. hesperus development and the

phenotypic rescue by 20-hydroxyecdysone are causal

proof of the involvement of LhSpot in ecdysteroid bio-

synthesis and related developmental processes, and

may provide an avenue for development of new con-

trol measures against Ly. hesperus.

Keywords: western tarnished plant bug, Lygus hes-

perus, Halloween gene, Spookiest, CYP307B1, RNA

interference.

Introduction

In insects, the fundamental processes of growth, devel-

opment and reproduction are coordinated by fluctuations

in the level of circulating ecdysteroids, the major forms

of which include ecdysone (E) and its derivative 20-

hydroxyecdysone (20E). During pre-adult development,

pulses of 20E trigger moulting and metamorphosis,

whereas in adult females, 20E functions in oogenesis,

vitellogenesis, choriogenesis and the early events of

embryogenesis (Morgan & Poole, 1977; Koolman, 1982;

Gilbert et al., 2002; Truman, 2005). Similar to vertebrate

steroidal biosynthesis pathways, insects utilize choles-

terol and/or plant sterols as ecdysteroid precursors

(Gilbert et al., 2002; Niwa & Niwa, 2011); however,

unlike vertebrates, insects lack genes such as squalene

synthase that are critical for synthesizing cholesterol

from simple precursor molecules (Gilbert et al., 2002;

Niwa & Niwa, 2011). Consequently, ecdysteroid biosyn-

thesis must rely on dietary supplied cholesterol

(obtained directly from prey by carnivorous insects) or

plant-derived phytosterols, which must first undergo

dealkylation to cholesterol in midgut cells (Gilbert et al.,

2002; Gilbert & Warren, 2005; Canavoso et al., 2001).

The absorbed cholesterol is transported via haemo-

lymph lipophorin to steroidogenic organs (prothoracic

glands in immatures and ovarian follicle cells in

adult females) where it is step-wise converted to E by a

series of conserved enzymatic reactions (Fig. 1). The

best-characterized genes in this pathway are the Hallow-

een genes, a suite of cytochrome P450 monooxygenases

First published online 18 May 2016.

Correspondence: J. Joe Hull, USDA-ARS Arid Land Agricultural Research

Center, 21881 N Cardon Lane, Maricopa, AZ 85138, USA. Tel.: 1 1 520

316 6334; fax: 1 1 520 316 6330; e-mail: [email protected]

E. Van Ekert and M. Wang contributed equally to this work.

550 Published 2016. This article is a U.S. Government work and is in the public domain in the USA

Insect Molecular Biology (2016) 25(5), 550–565 doi: 10.1111/imb.12242

initially identified in Drosophila melanogaster develop-

mental mutants (J€urgens et al., 1984; N€usslein-Volhard

et al., 1984; Wieschaus et al., 1984).

The initial step in the ecdysteroid biosynthetic path-

way, conversion of cholesterol to 7-dehydrocholesterol,

is mediated by a Rieske-domain oxygenase termed

Neverland that catalyses 7,8,-dehydrogenation of the

cholesterol precursor. The steps converting the

7-dehydrocholesterol into 5b-ketodiol (2,22,25-trideox-

yecdysone) involve multiple enzymatic steps currently

known to be mediated by a short-chain dehydrogenase

termed Shroud, a P450 monooxygenase(s) CYP307A1/

A2 termed Spook/Spookier, and a CYP6T3 monooxy-

genase (Iga & Kataoka, 2012; Niwa & Niwa, 2014).

Although D4-ketodiol and diketol have been identified as

likely intermediate substrates, additional substrates and

the precise nature of the reactions that yield 5b-ketodiol

have yet to be fully elucidated. Consequently, these

steps are frequently referred to as the ‘black box’ of

ecdysteroid biosynthesis (Namiki et al., 2005; Ono et al.,

2006; Rewitz et al., 2007). 5b-ketodiol is further hydroxy-

lated to yield 5b-ketotriol (2,22-dideoxyecdysone), 2-

deoxyecdysone and E by the Phantom, Disembodied

and Shadow P450 monoxygenases, respectively. E is

then converted in peripheral tissues into the active hor-

mone, 20E, by the P450 monoxygenase Shade.

Given the indispensable role of ecdysteroids in regu-

lating and coordinating the critical insect processes of

growth, development and reproduction, the lack of func-

tional redundancy amongst the Halloween suite of genes

is surprising. Identification or prediction of Halloween

genes in diverse insect species (Namiki et al., 2005;

Ono et al., 2006, 2012; Sztal et al., 2007; Christiaens

et al., 2010; Iga & Smagghe 2010; Marchal et al., 2010;

Yamazaki et al., 2011; Hentze et al., 2013; Jia et al.,

2013a,b; Luan et al., 2013; Pondeville et al., 2013; Zhou

et al., 2013; Shahzad et al., 2015), as well as other

arthropods (Rewitz & Gilbert, 2008; Cabrera et al.,

2015), has revealed that each gene is more similar to its

respective orthologues than to other P450 monoxyge-

nases, suggesting the specific steps in ecdysteroid bio-

synthesis are catalysed by a single enzyme. Paralogues,

however, have been identified for the CYP307 P450

monoxygenases, which have undergone lineage-specific

duplications/losses, with three paralogues CYP307A1

(Spook, Spo), CYP307A2 (Spookier, Spok) and

CYP307B1 (Spookiest, Spot) identified in various spe-

cies, albeit with only two of the three present in any one

species (Rewitz et al., 2007; Sztal et al., 2007; Rewitz &

Gilbert, 2008). In Dr. melanogaster, duplication of the

ancestral Spok gene, thought to be specific to the Dro-

sophilidae, probably yielded Spo. Spok-like sequences,

however, have recently been annotated in Chilo suppres-

salis (AHW57298) (Wang et al., 2014), Cnaphalocrocis

medinalis (AJN91167), Dendroctonus armandi

(ALD15893) (Dai et al., 2015), Nilaparvata lugens

(AIW79977) and Laodelphax striatellus (AGU16448) (Jia

et al., 2015), with RNA interference (RNAi)-based knock-

down in the last species reportedly supporting a func-

tional role in development. The third paralogue, Spot, is

thought to be a lineage-specific duplication of Spo. To

date, Spot-like sequences have been identified/predicted

in Aedes aegypti (Rewitz et al., 2007), Anopheles gam-

biae (Rewitz et al., 2007) and Tribolium castaneum

(Rewitz et al., 2007; Christiaens et al., 2010). In Apis

mellifera, Spot appears to be the only CYP307

paralogue (Rewitz et al., 2007; Yamazaki et al., 2011).

This paralogue reduction is not characteristic of the

Hymenoptera, as the lone CYP307-like sequence in

Nasonia vitripennis shares greater homology with

Spo than Spot (Rewitz et al., 2007). Initial analysis of

the Acyrthosiphon pisum genome suggested the pres-

ence of three Spo-like sequences (Ap-Spo1–3;

XM_001945726, XM_001946260 and XM_001948680)

Figure 1. Current model of the ecdysteroid biosynthetic pathway in insects.

RNAi knockdown of Spookiest in Lygus hesperus 551

Published 2016. This article is a U.S. Government work and is in the public domain in the USA, 25, 550–565

with the first two genes proposed as Spo orthologues

and the third as a Spot orthologue (Christiaens et al.,

2010). Subsequent genome annotations have collapsed

the Spo paralogues into a single gene.

Whereas the molecular basis of ecdysteroid biosyn-

thesis has been extensively characterized in holometab-

olous insects, our understanding of this pathway in

hemimetabolous insects is limited to a few species –

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MF F L V T F S V T - - - - - - - - V F L V F V L S GL E R L K S V R K F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K E I AP GP R P WP L I GS L HL MGGS - S S P F QV F T K L S K T Y GS I Y S I T L GS S S C V V V NS F NL I K K V L I S K GGH

F GGR P DF - - - - - - - I R F HK L F GGDR NNS L AL C DWADL QK T R R S I AR L Y C S P R F AS T NY DR L R E V GL QE MNE F I C E L S NI T T K S - - HDV E L K P L I MAT C ANMF T S Y MC S T - - - - - - - - - R F N- Y DDK E F T K T V L L F DNI F WE I NQGY AV DF L P

WL L P F Y NR HMNR L S K WS K DI R QF I L S R I I DHHR S - - - - - - - - - - - T L D- - - - - - - P NAP P R DF T DAL L L HL E E - - - - - - - - - - - - - - - - - - DE L L NWQHI I F E L E DF L GGHS AI GNL V ML AL AGI V K NP E V GT K I QE E I DR V V - G- NC R P V D

L F DK P DMP F T E AT I L E T L R MS S S P I V P HV ANQDT MI E GY F V K K GS V V F L NNY E L NT GE K Y WS DP K K F K P E R F I I N- - - - - - - - - - - - - - - - - - - - - - - - - - - - - NQI MK - - - - - - - - - - - - - - - P E F F I P F S T GK R T C I GQR L V QGF S F I L L

AT I L QNY NV S C S DL S S I H- F QP AC V AL P P S T F S L NL K P R T I QQE Q K G

I V V S V L - - - - - - - - C V I V L V L L V K L WAT K R R K P - - - - - - - - - - - - - - - - - - - - - - - - - - - - K S AK P I P GP E P WP L I GS L HL L GK Y - E T P F E GF T AL S K I Y GDI F S I T MGS T P C V V V NNF DL I K E V L I T K GGD

F GGR P DF - - - - - - - MR F HK L F GGDR NNS L AL C DWS AL QK T R R S I AR T Y C S P R F T S L QY AT V NT V GANE L DAF MAE MQNHP L NT - - - P L DI K P V V L AAC ANMF L QY MC S T - - - - - - - - - S F P - Y DDQQF K K V V R DF DE I F WE I NQGY AV DF L P

WL L P MY S K HMS K L S QWAADI R E F I L AR I V QDHV N- - - - - - - - - - - T L D- - - - - - - Y HAP P R DF T DAL L I HL R E - - - - - - - - - - - - - - - - - - DP NME WNHI I F E L E DF I GGHS AV GNL V MMI L AS V V K Y P E V AK K I QE E V DE V T - R - GQR Y V N

L F DK AAMP Y S E AV L WE T L R K GS S P I V P HV AS T DT E I E GY P V K R GT MI F L NNY E L NL S P QY WDE P E K F K P E R F L S P A- - - - - - - - - - - - - - - - - - - - - - - - - - - - GNV V K - - - - - - - - - - - - - - - P T HF I P F S T GK R AC I GQR L V QC F S F I I V

S T L L QNY NI S P - - GS E I K - I K P GC V AV P P DC F K V I L T P R NR K S D

MC L K MMT F GL AK C T I T GV DGHGP R DGGT P I L L V AGAAT GHL QANL QI AAAP P DML AL L C L C V V L L V WWF S R P K K S P S T I P GP R P WP L I GS MHL L AGH- E T P F QAF T AL S R V Y GDI F S I HL GS AS C V V V NNF K L I K E V L I AK GGD

F GGR P DF - - - - - - - AR F HK L F GGDR NNS L AL C DWS S L QK T R R S I AR T Y C S P R F T S L QY DR V NNV GE E E L K S F L HQL DQL P HGQ- - - P C NV K P AV L MV C ANMF T QY MC S T - - - - - - - - - S F A- Y E DK GF QK I V R Y F DE I F WE I NQGY AV DF L P

WL L P V Y T GHMK K I S NWAT E I R QF I L S R I I DK HR A- - - - - - - - - - - T L D- - - - - - - T NS P P R DF T DAL L MHL E E - - - - - - - - - - - - - - - - - - DP NMNWQHI I F E L E DF L GGHS AI GNL V MV T L AAV V DHP E V AK R I QE E V DQV T - G- GT R C P N

L F DK AAMP Y T E AT I L E T L R T AS S P I V P HV AS K DT E I DGHE V S K GT I V F I NNY E L NQGDAY WDE P GL F K P E R F L S S T - - - - - - - - - - - - - - - - - - - - - - - - - - - - GNI V K - - - - - - - - - - - - - - - P AHF I P F S T GK R T C I GQR L V QC F S F V V L

AT L L QY Y DV S T K - - E S V K - V QP GC V AV P P DC F K L V L T P R K

MS S L I I V L F V F - - - - - - - - AL AV Y K L L R R K T V R WV K T N- - - - - - - - - - - - - - - - - - - - - K Y GGV E T AI L R T A P GP V C WP I I GS L HL L GGH- E S P F QAF T E L S K K Y GDI F S V K L GS ADC V V V NNL S L I R E V L NQNGNV

V AGR P DF - - - - - - - L R F HK L F AGDR NNS L AL C DWS NL QL R R R NL AR R HC GP K QHT DS Y AR I GT V GT F E S V E L I QT L K GL T S R S - DAS I DL K P I L MK S AMNMF S NY MC S V - - - - - - - - - R F D- DE DL E F QK I V DHF DE I F WE I NQGY AV DF L P

WL AP F Y K K HME K L S NWS QDI R S F I L S R I V E QR E I - - - - - - - - - - - NL D- - - - - - - T E AP E K DF L DGL L R V L HE - - - - - - - - - - - - - - - - - - DP T MDR NT I I F ML E DF L GGHS S V GNL V ML C L T AV AR DP E V GR K I R QE I DAV T - R - GK R P V G

L T DR S HL P Y T E AT I L E C L R Y AS S P I V P HV AT E NANI S GY GI E K GT V V F I NNY V L NNS E QY WS E P E K F DP S R F L E K T - - - - - - - R V R T R R NS QC DS GL E S DS E R - AP V GK - - P DV E R E ML S V K K NI P HF I P F S I GK R T C I GQT MV T S MS F T MF

ANI MQS F E V GV E NI NDL K - QK P AC V AL P K NT Y K MHL I P R K

ML S AL F L L AAF - - - - - - - - L I AV Y K I Y F S NT I T Y K K I T - - - - - - - - - - - - - - - - - - - - - K Y GE NK T L V L K E A P GP T P L P I I GNL HL L GK H- E S P F QS F T E L AK K Y GDI F S L QL GT AK C L V V NNL DL I R E V L NQNGK F

F GGR P DF - - - - - - - I R F HQL F AGDR NNS L AL C DWS NL QL R R R NL AR R HC GP K QHT DNF AR I GDV AT F E S I E L L QT L K NI T R T S - DS S I NL K P I L MT T AMNMF S HY MC NV - - - - - - - - - R F DAE NDE DF K R I V DHF DE I F WE I NQGY AL DF L P

WL QP F Y K K HL DK L S NWS ADI R S F I L S R I V E QR E L - - - - - - - - - - - DL D- - - - - - - V E GP E K DF L DGL L K V L HE - - - - - - - - - - - - - - - - - - DP T V DR NT I I F ML E DF L GGHS S V GNL V ML C L T AV AR DP E V GR K V R AE L DAL T - K - GK R P V T

L L DR HS L P Y T E AT V L E C L R Y AS S P I V P HV AT E NAAI S GY GV E K GT I V F I NNY E L NT S E E Y WQE P E K F DP T R F L E K T - - - - - - - K V R V R R NS F C DS GME S DGE R P S K P S E - - - - T E K E V WS V K R NI P HF L P F S I GK R T C I GQT L V T T MS F V MF

AS I MQE F E I GAE S L E DL R - QK P AC V AL P K DT Y NL F L I P R K H

MS AI L L AV V I V L - - - - - - - - I AY K F L L P R K NT V T L K R V N- - - - - - - - - - - - - - - - - - - - - - R T GE V I NT L R T A P GP T P L P I I GNL HL L GK H- E S P F QAF T E L S K QY GDI F S L K L GNT P C L V V NNL E L I R E V L NQNGK F

F GGR P DF - - - - - - - I R F HK L F AGDR NNS L AL C DWS NL QT R R R NMAR R HC GP K QHT DNF T R I GS V AT F E S I DL I QT L R S I ANT T - E S S I NL K P ML MS T AMNMF C HY MC NV - - - - - - - - - R F DP E NDV E F K K I V DHF DE I F WE I NQGY AV DF L P

WL E P L Y K K HMDK L S GWS QDI R T F I L S R I V E QR E M- - - - - - - - - - - S L D- - - - - - - V E GP E K DF L DGL L R V L HD- - - - - - - - - - - - - - - - - - DP S V DR NT I I F ML E DF L GGHS S V GNL V ML C L AAV AR DP E V GK K I K AE I DGHT - K - GK R AI T

L S DR S S L P F T E AT I L E C L R Y AS S P I V P HV AT E NAS V AGF GV E K GT V V F I NNY E L NT S E QY WR E P E K F DP S R F L E R T - - - - - - - K I R V R R NS HC DS GME S DS E R T GP I DK - AT E T E K E V V S V R K NI P HF L P F S I GK R T C I GQT L V T T MS F V MF

AS I MQE F DV AAE NL E DL R - QK P AC V AL P K DT F NL Y L V P R K H

MS AI L L V AI - - - - - - - - V AL C V Y K F F S R K V T F K S GV - - - - - - - - - - - - - - - - - - - - - - - - - - - - E K V AP AP GP V P L P V I GNL HL L GK H- E S P F QAF T E L S K T Y GDI F S L K L GNT P C L V V NNL E L I R E V L NQNGK F

F GGR P DF - - - - - - - I R F HK L F AGDR NNS L AL C DWS S L QT R R R NL AR R HC GP K QHP E NF AR I GS V AT F E S I E L L QT L R T I AT T T - AS P I NL K P I L MAT AMNMF S HY MC NI - - - - - - - - - R F E - E S DP E F R K I V DHF DE I F WE I NQGY AV DF L P

WL E P L Y K K HMDK L S GWS QDI R T F I L S R I V E HR E M- - - - - - - - - - - NL E - - - - - - - T DGP E K DF L DGL L R V L K D- - - - - - - - - - - - - - - - - - DS DV DR NT I I F ML E DF L GGHS S V GNL V ML C L AAMAR DADV AK R V K AE I DGV T - K - GK R AV T

L S DR S S L P Y T E AT V L E C L R Y AS S P I V P HV AT E NAV I S GY GV E K GT V V F I NNY E L NT S E QY WK E P E K F DP S R F L E K S - - - - - - - K I R V R R NS QC DS GME S DGE R S GP L NK - AT E ME K E V T S V R K NI P HF L P F S I GK R T C I GQT L V T T MS F V MF

AS I MQE F DV AAE NI E DL R - QK P AC AAL P K DT F S L Y L I P R K H

MAY T GL I L AV L T - - - - - - - I I L S V V C Y F K I L Y E WHR K V R I QT V R P S R L QK L AT AAT T L P QQS T T E L MT F P QA P GP V P WP I L GS L AF L GQY - DV P F E GF T AL AK K Y GDL Y S I T L GS T R C L V V NNL DL I R E V L NQNGR Y

F GGR P DF - - - - - - - L R F HK L F GGDR NNS L AL C DWS T L QQK R R NL AR K HC S P S DAS S Y Y QK MS DV GV ME MHR F MDQL DE V V C P G- - K DF K V K P L I MQAC ANMF S E Y MC S V - - - - - - - - - R F D- Y NDQGF MS I V R NF DE I F WE I NQGY AV DF L P

WL AP F Y HK HMS K L T R WS AE I R DF I L E R I V NE R E Q- - - - - - - - - - - T L G- - - - - - - DDDT E R DF ADAL L K S L R L - - - - - - - - - - - - - - - - - - DP S V T R DT I MY ML E DF L GGHS AI GNL V ML AL GY I AK HP E V GHR I QR E I DR I T E R - GK R NV T

L Y DT E S MP Y T V AT I F E V L R Y S S S P I V P HV AT E DT C I AGY GV T K GT V V F I NNY E L NT S E R Y WS E P K R F NP S R E NE R T - - - - - - - - - - - - - - - - - - - - - - - - - - - - L QI E R - - - - - - - - - - - V R K NI P HF L P F S I GK R T C I GQNL V R GF S F I I I

ANI L QK Y DV HS NDL S L I K - MY P AC V AV P P DT Y P L AF T QR S T V AA

ML T S V F Y V L F AI A- - - - - - - - I T I I L I S Y V F L L L K C K QK A- - - - - - - - - - - - - - - F V V I GL L Y QE K K Y QC F DQA P GP HP WP I I GNI NL L GR F QY NP F Y GF GT L T K K Y GDI Y S L S L GHT R C I V V NNV DL I K E V L NK NGK Y

F GGR P DF - - - - - - - F R Y HK L F GGDR NNS L AL C DWS QL QQK R R NL AR R HC S P R E S S S Y F S K MS E I GGL E V NQL L DQL T NI S S GY - - - P C DV K P L I L AAS ANMF C QY MC S V - - - - - - - - - R F N- Y S DK GF QK I I E Y F DE I F WE I NQGY S F DY I P

WL V P F Y C NHI S R I V HWS AS I R K F I L E R I V NHR E S - - - - - - - - - - - NI N- - - - - - - I NE P DK DF T DAL L K S L K E - - - - - - - - - - - - - - - - - - DK NV S R NT I I F ML E DF I GGHS AV GNL V ML AL AY I AK NP T I AL HI R NE V DT V S AK - GI R R I C

L Y DMNV MP Y T MAS I S E V L R Y S S S P I V P HV AME DT V I K GF GV R K GT I V F I NNY V L NMS E S F WNHP E QF DP E R F L E NNF T NNK E S GL K C DDNK R T E F I R K NDNDGS T K S K K Y GK QNL NNK L - L K K S I P QF L P F S V GK R T C I GQS L V R GF GF L L L

ANI I QNY NV NS ADF S K I K - L E K S S I AL P K K C F K L S L R P R T

ML AAL I Y T I L AI L L S V L - - - - - - - - AT S Y I C I I Y GV K R R V L QP V - - - - - - - - - - - - - - - K T K NS T E I NHNAY QK Y T QA P GP R P WP I I GNL HL L DR Y R DS P F AGF T AL AQQY GDI Y S L T F GHT R C L V V NNL E L I R E V L NQNGK V

MS GR P DF - - - - - - - I R Y HK L F GGE R S NS L AL C DWS QL QQK R R NL AR R HC S P R E F S C F Y MK MS QI GC E E ME HWNR E L GNQL V P G- - E P I NI K P L I L K AC ANMF S QY MC S L - - - - - - - - - R F D- Y DDV DF QQI V QY F DE I F WE I NQGHP L DF L P

WL Y P F Y QR HL NK I I NWS S T I R GF I ME R I I R HR E L - - - - - - - - - - - S V D- - - - - - - L DE P DR DF T DAL L K S L L E - - - - - - - - - - - - - - - - - - DK DV S R NT I I F ML E DF I GGHS AV GNL V ML V L AY I AK NV DI GR R I QE E I DAI I E E - E NR S I N

L L DMNAMP Y T MAT I F E V L R Y S S S P I V P HV AT E DT V I S GY GV T K GT I V F I NNY V L NT S E K F WV NP K E F NP L R F L E P S - - - - - - - - - - - - - - - - - - - - - K E QS P K NS K GS DS GI E S DNE K L QL K R NI P HF L P F S I GK R T C I GQNL V R GF GF L V V

V NV MQR Y NI S S HNP S T I K - I S P E S L AL P ADC F P L V L T P R E K I GP L

ApSpot PhSpot AmSpot LhSpot TcSpot AgSpot LsSpot NlSpot ApSpo SfSpo NlSpok PhSpo DaSpo TcSpo BmSpo HaSpo CsSpok CmSpok AgSpo DmSpok DmSpo

heme-binding domainPERF domainhelix K

helix I

helix C

P/G rich region





I

552 E. Van Ekert et al.


Ac. pisum (Christiaens et al., 2010), Bemisia tabaci

(Luan et al., 2013), La. striatellus (Jia et al., 2013a,

2014, 2015; Wan et al., 2014a,b) and Sogatella furcifera

(Jia et al., 2013b; Wan et al., 2014c). Excluded from this

list are mirid plant bugs, many of which are polyphagous

agricultural pests (Wheeler, 2001; Schuh, 2013). One

genus of this group that is of particular economic impor-

tance is Lygus. Traditionally managed with broad-

spectrum insecticides, reports of insecticide resistance

in field populations (Snodgrass, 1996; Snodgrass &

Scott, 2002; Snodgrass et al., 2009) underscore the

need for alternative control tactics. One promising area

for potential development is targeting the molecular

machinery that regulates developmental and reproduc-

tive processes, such as those controlled by ecdyste-

roids. A generally poor understanding of the underlying

physiology and limited molecular resources have ham-

pered efforts to devise such approaches for Lygus.

Recent transcriptome assemblies (Hull et al., 2013,

2014) for the western tarnished plant bug (Lygus hespe-

rus Knight), the dominant pest Lygus species in the

western USA (Schwartz & Footit, 1998), have however

greatly facilitated gene identification and annotation

processes and have opened the possibility of exploring

gene functionality through the use of RNAi-mediated

knockdown. Furthermore, we recently demonstrated the

criticality of ecdysteroid timing in controlling the

nymphal–adult moult in Ly. hesperus (Brent et al., 2016).

In this study, we build on those findings and report

transcriptome-based identification of a single Spo-like

sequence that shares significantly greater sequence

homology with Spot orthologues than with either Spo or

Spok. The transcript is expressed in select adult tissues

and throughout development, with transcript abundance

fluctuating inversely with previously reported ecdysteroid

levels. RNAi-mediated knockdown and subsequent 20E

rescue confirm the importance of the LhSpot transcript

in controlling the timing of adult eclosion and develop-

ment of adult characteristics. These are important initial

steps toward developing control approaches that target

a potential vulnerability of this key pest.

Results

Identification and phylogenetic characterization

of LhSpot

To identify the first cytochrome P450-catalysed step in Ly.

hesperus ecdysteroid biosynthesis (ie CYP307), we per-

formed a tBLASTn search of our recent transcriptome

assembly (Hull et al., 2014) using queries consisting of

representative Spo sequences from five insect orders:

Diptera (Dr. melanogaster), Lepidoptera (Bombyx mori),

Hymenoptera (Na. vitripennis), Coleoptera (T. castaneum)

and Hemiptera (Ac. pisum and La. striatellus). All of the

queries generated the same unigene hit with an e-val-

ue<102127. The next closest unigene hits had e-values

ranging between 10232 to 10246, none of which exhibited

homology with the Halloween genes, suggesting the pres-

ence of a single Spo-like sequence in our transcriptome

assembly. Multiple Spo paralogues (ie Spo, Spok and

Spot), however, have been reported in various species

from multiple insect orders (Rewitz et al., 2007). The pres-

ence of a single sequence in Ly. hesperus may be the

result of exclusion of temporally or spatially restricted tran-

scripts (eg transcripts specifically regulated in develop-

ment) in our transcriptome assembly, or alternatively may

reflect loss of those genes.

Using cDNA generated from fifth instars 2 days after

the moult and primers designed to the putative Spo-like

open reading frame (ORF), we amplified a 1482-

nucleotide (nt) product that encodes a 493-amino acid

protein with a predicted molecular weight of 56.1 kDa.

The unigene ORF shares considerable identity (54.7%)

with a putative Spo orthologue in the hemipteran pest,

La. striatellus (AGI92303), but only moderate identity

(�37%) with Spo orthologues in Dr. melanogaster

(AAQ05973), Bo. mori (BAM73857) and Ac. pisum

(XP_001945761). Despite relatively low sequence iden-

tity, the Ly. hesperus sequence contains motifs charac-

teristic of insect cytochrome P450s (Werck-Reichhart &

Feyereisen, 2000) including a heme-binding loop

(PFxxGxRxCxG), helix K (ExxR) and an aromatic PERF

Figure 2. Multiple sequence alignment of Lygus hesperus Spookiest (LhSpot) with Spook paralogues from 14 insect species. Sequences were aligned using

MUSCLE (Edgar, 2004) with default settings. Amino acids highlighted in black indicate amino acid identity whereas those in grey represent amino acid

conservation. Characteristic P450 domains/motifs (Pro/Gly rich region, helix K, Pro-Glu-Arg-Phe (PERF) domain and heme-binding domain) are indicated. The helix

C and helix I motifs (dashed lines with grey font) do not meet conventional definitions for these regions (ie helix C 5 WXXXR; helix I 5 GxE/DTT/S) but correspond

to regions defined as such in reports of insect CYP307A1 orthologues (Iga & Smagghe, 2010; Jia et al., 2015; Shahzad et al., 2015). Amino acid substitutions spe-

cific to Spot paralogues that are located near the putative helix C are indicated with closed ovals, those near helix I with open squares and miscellaneous spot spe-

cific changes are indicated with open ovals. Residues comprising the Ly. hesperus Spot hydrophobic targeting sequence are outlined. Abbreviations: ApSpot,

Acyrthosiphon pisum Spookiest (XP_001948715); PhSpot, Pediculus humanis corporis Spookiest (XP_002425350); AmSpot, Apis mellifera Spookiest

(BAJ54121); LhSpot, Ly. hesperus Spookiest; TcSpot, Tribolium castaneum Spookiest (EFA05673); AgSpot, Anopheles gambiae Spookiest (AAV28189); LsSpot;

Laodelphax striatellus Spookiest (AGI92303); NlSpot, Nilaparvata lugens Spookiest (AIW79978); ApSpo, Ac. pisum Spook (XP_001945761); SfSpo, Sogatella fur-

cifera Spook (AGU16443); NlSpok, Ni. lugens Spookier (AIW79977); PhSpo, Pe. humanis corporis Spook (XP_002429996); DaSpo, Dendroctonus armandi Spook

(ALD15893); TcSpo, T. castaneum Spook (EFA11558); BmSpo, Bombyx mori Spook (BAM73857); HaSpo, Helicoverpa armigera Spook (AID54856); CsSpok,

Chilo suppressalis Spookier (AHW57298); CmSpok, Cnaphalocrocis medinalis Spookier (AJN91167); AgSpo, An. gambiae Spook (AHB59617); DmSpok, Dro-

sophila melanogaster Spookier (EDP28053); DmSpo, Dr. melanogaster Spook (AAQ05973).



domain (PxxFxPE/DRF) (Fig. 2). Although the consen-

sus sequences for two additional P450 motifs, helix C

(WxxxR) and helix I (A/GGxD/ETT/S), have diverged

from that seen in the other Halloween proteins (Namiki

et al., 2005; Ono et al., 2006; Iga & Smagghe, 2010;

Marchal et al., 2011; Zhou et al., 2013; Cabrera et al.,

Tc (X

P_9

7425

2)

Ap (X

P_00

1948

299)

37

Nv (X

P_00

1601

675)

59

Dm (NP_5

2481

0)

44

Bm (NP_001036953)

100 Tc (NP_001123894)

Nv (NP_001166018)58

Ap (XP_008187090)

Dm (AAQ05972)Bm (NP_001106219)

75

54

100

91

Tc (XP_008193656)

Bm (NP_001106224)

Dm (AAL86019)

41

Ap (XP_001944183)

Nv (X

P_001607634)

48

26

100

Dm

(NP_

5733

19)

Bm (B

AD34

476)

67

Nv (XP_0

0821

4357

)

49

Tc (XP_968477)

85Ap (XP_001947874)

100Ap (XP_001948715)

LhSpo-like

61

Tc (XP_974280)

99

Nv (XP_001603435)

Bm (NP_001104833)

Dm (AAQ05973)

Dm

(NP_001104460)

9998

91

100

100

Tc - Tribolium castaneumNv - Nasonia vitripennisDm - Drosophila melanogasterBm - Bombyx moriAp - Acyrthosiphon pisum

Spook/Spookier/Spookiest(CYP307A1/A2/B1)

Phantom(CYP306A1)

Disembodied(CYP302A1)

Shade(CYP314A1)

Shadow(CYP315A1)

A

B

Spo/SpokCYP307A1/CYP307A2

SpotCYP307B1

Bo. mori Spo (BAM73857)

H. armigera Spo (AID54856)

Ch. suppressalis Spok (AHW57298)

Cn. medinalis Spok (AJN91167)

An. gambiae Spo (AHB59617)

Dr. melanogaster Spok (EDP28053)

Dr. melanogaster Spo (AAQ05973)

Ac. pisum Spo-1 (XP_001945761)

De. armandi Spok (ALD15893)

So. furcifera Spo (AGU16443)

Ni. lugens Spok (AIW79977)

T. castaneum Spo (EFA11558)

Ac. pisum Spo-3 (XP_001948715)

P. humanus Spot (XP_002425350)

LhSpot

T. castaneum Spot (EFA05673)

An. gambiae Spot (AAV28189)

Ap. mellifera Spot (BAJ54121)

La. striatellus Spo (AGI92303)

Ni. lugens Spot (AIW79978)98

65

58

99

97

89

36

44

21

49

65

9

77

9724

42

21

17

0.1

P. humanus Spo (XP_002429996)



2015; Jia et al., 2015; Shahzad et al., 2015), the regions

associated with these motifs are well conserved in the

LhSpo-like sequence and orthologues in other species

(Fig. 2).

To further confirm annotation of the unigene sequence,

we examined its phylogenetic relationship with Halloween

proteins in Dr. melanogaster, Bo. mori, Na. vitripennis, Ac.

pisum and T. castaneum (Fig. 3A). Consistent with the

sequence analyses and the initial annotation, the unigene

sequence aligned to the Spo clade, with highest similarity

(as expected given the shared hemimetabolous lineage)

to Ac. pisum Spo. To increase the phylogenetic resolution,

we limited the analysis to 20 sequences annotated as

Spo, Spok or Spot. When aligned with this larger clan-

specific data set, sequence conservation between the

unigene and the Spo/Spok orthologues averaged 38.5%,

whereas in the Spot group identity was 48.6% (Table S1),

suggesting that the LhSpo-like sequence is a Spot ortho-

logue. Phylogenetic analyses (maximum likelihood, neigh-

bour joining, minimum evolution and the Unweighted Pair

Group Method with Arithmetic Mean method (UPGMA))

further support this designation as the LhSpo-like

sequence clustered away from Spo and Spok in a largely

Spot-specific clade (Fig. 3B). The lone exception to that

clade is a sequence from La. striatellus (AGI92303) anno-

tated as CYP307A1; however, given the phylogenetic

relatedness with Spot we suggest that it has been misan-

notated in the database and that the La. striatellus

sequence is actually a Spot orthologue. Based on these

phylogenetic relationships and shared sequence similar-

ities, we have annotated the LhSpo-like sequence as a

Spot orthologue (LhSpot; GenBank accession

KT818622).

Heterologous expression of a LhSpot fluorescent

chimera in cultured insect cells

Spo homologues in Dr. melanogaster and Bo. mori co-

localize in Drosophila S2 cells with markers of the

endoplasmic reticulum (ER)/secretory pathway (Namiki

et al., 2005; Ono et al., 2006). Cursory domain analy-

ses (hydrophobic amino terminus with Pro/Gly-rich

region), homology with microsomal cytochrome P450s

and subcellular localization prediction algorithms (WoLF

PSORT and Target P1.1) suggest that LhSpot also

localizes to the ER/secretory pathway. To provide fur-

ther insights into the presumptive subcellular localiza-

tion of LhSpot, we co-expressed fluorescent chimeras

of LhSpot (LhSpot-Venus) and the human KDEL (Lys-

Asp-Glu-Leu) endoplasmic reticulum protein retention

receptor (HsKDELR-mCherry), a marker of the ER/

secretory pathway (van der Vlies et al., 2002; Maro-

niche et al., 2011), in cultured Trichoplusia ni cells.

Cells expressing LhSpot-Venus exhibited a diffuse

green fluorescent pattern typical of ER that extensively

overlaid with the HsKDELR-mCherry red fluorescent

signal (Fig. 4), suggesting co-localization of the two

proteins. Although this cellular localization is consistent

with previous studies and suggests that LhSpot resides

Figure 3. Phylogenetic relationships of the putative Lygus hesperus Spookiest (LhSpot) sequence. (A) Phylogenetic analysis using full-length Halloween

protein sequences from five insect species. The respective sequences were aligned using MUSCLE (Edgar, 2004) and the evolutionary history was inferred

using the maximum likelihood method implemented in MEGA6 (Tamura et al., 2013). The bootstrap consensus tree is based on 1000 replicates with support

values indicated at the branch points. Accession numbers of the sequences used in the analysis are indicated in parentheses and can be found in Table S3.

(B) Phylogenetic relationships amongst the CYP307 group of Halloween genes. The analysis was performed as above using the CYP307A1-like sequences

from Fig. 2. The evolutionary history was inferred using the maximum likelihood method based on the Jones Thornton-Taylor (JTT) matrix-based model. The

tree with the highest log likelihood (21496.1948) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the

branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Sequence accession numbers are indicated in

parentheses. Species abbreviations are: Ac. pisum, Acyrthosiphon pisum; An. gambiae, Anopheles gambiae; Ap. mellifera, Apis mellifera; Bo. mori, Bombyx

mori; Ch. suppressalis, Chilo suppressalis; Ch. medinalis, Cnaphalocrocis medinalis; De. armandi, Dendroctonus armandi; Dr. melanogaster, Drosophila

melanogaster; H. armigera, Helicoverpa armigera; La. striatellus, Laodelphax striatellus; Ni. lugens, Nilaparvata lugens; P. humanus, Pediculus humanus

corporis; So. furcifera, Sogatella furcifera; T. castaneum, Tribolium castaneum.

LhSpot-Venus HsKDELR-mCherry MergeNucBlue

Figure 4. Subcellular localization of a fluorescent Lygus hesperus Spookiest (LhSpot) chimera in cultured insect cells. Cells were co-transfected with expression

plasmids encoding LhSpot-Venus (green) and an endoplasmic reticulum marker (ER), Homo sapiens KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein reten-

tion receptor-mCherry (KDELR-mCherry; red). At 48 h post-transfection, cells were labelled with the nuclear marker NucBlue (blue) and live cells were imaged on a

confocal scanning laser microscope using a 603 water objective. Overlay of the green and red fluorescent signals (yellow in the merge panel) suggests co-

localization of LhSpot-Venus with KDELR-mCherry in the ER. Scale bar 5 10 mm.



in the ER/secretory pathway, in vivo confirmation of this

finding in native tissues remains to be demonstrated.

Reverse Transcription-PCR (RT-PCR) expression profile

of LhSpot

Spo orthologues have been reported to be expressed

throughout embryonic and larval development as well as

in select adult tissues (Rewitz et al., 2007). We used RT-

PCR to examine the temporal and spatial abundance of

LhSpot transcripts. LhSpot transcripts were amplified

from eggs and throughout nymphal development (Fig.

5A). To determine the potential role of LhSpot abun-

dance in relation to the nymphal–adult moult, we exam-

ined expression at 24-h intervals from the initiation of

the fifth instar until 48 h post-adult eclosion. LhSpot tran-

scripts exhibited rhythmic expression, with levels peaking

at 1 and 3 days after moulting to the fifth instar. Tran-

scripts were barely detectable on the day of eclosion to

the fifth instar and were completely undetectable in

adults until 48 h post-eclosion (Fig. 5A). LhSpot was

amplified from all three nymphal segments, but was spa-

tially restricted in adults (Fig. 5B). In males, transcripts

were amplified from bodies and to a lesser extent heads

with no detectable amplification from male reproductive

tissues (testes and accessory glands). In females,

amplimers were detectable from head, bodies and repro-

ductive tissue (ovary and seminal depository), albeit at

varying abundances (Fig. 5B).

RNAi-mediated knockdown of LhSpot

To examine the functional role of LhSpot, we injected

double-stranded RNAs (dsRNAs) corresponding to either

a 547-bp fragment (nt 846–1352) of LhSpot or to the

complete enhanced green fluorescent protein (EGFP)

ORF into newly eclosed fifth instars. RT-PCR analysis of

transcript levels at 48 h post-injection revealed reduced

expression of LhSpot in the LhSpot dsRNA-injected

nymphs compared with the non-injected and EGFP

dsRNA-injected controls with no discernible effects on

expression of the control genes actin and glyceralde-

hyde 3-phosphate dehydrogenase (GAPDH) (Fig. 6A).

Because a reduction in LhSpot expression is expected

to negatively affect ecdysteroid biosynthesis and thus

ecdysone titres, we examined the effects of LhSpot

knockdown on the expression of E74 and E75, two

ecdysone-inducible transcription factors (Thummel,

1996), as a proxy for measuring circulating ecdysone.

Transcript levels of E74 and E75 were both reduced in

nymphs injected with LhSpot dsRNA compared with

controls (Fig. 6A), strongly suggesting that LhSpot

knockdown had impacted ecdysteroid levels. We next

examined the effects of LhSpot knockdown on adult

development. At 3 days post-injection, we detected no

noticeable phenotypic difference between LhSpot

dsRNA-injected and control nymphs (Fig. 6B). However,

consistent with a disrupted ecdysteroid signalling path-

way, LhSpot dsRNA-injected nymphs failed to undergo

adult eclosion and maintained a nymphal appearance

(absence of wings and smaller size), albeit with elon-

gated abdomens and black cuticular banding on the dor-

sum, even at >20 days post-injection (Fig. 6B).

Surprisingly, despite the nymphal appearance, LhSpot

dsRNA-injected nymphs at 25 days post-injection exhib-

ited limited oogenesis producing a few stage 3 oocytes

(Spurgeon & Brent, 2010) that are typically observed in

young adult females (Fig. 6C).

More comprehensive analysis of the effects of LhSpot

knockdown on the timing of adult development showed

that eclosion typically occurred 3–4 days after treatment

in non-injected, buffer-injected and EGFP dsRNA-

injected nymphs, but was never achieved within the time

frame (25 days post-injection) of our study (Fig. 7). Fur-

thermore, nymphs injected with LhSpot dsRNA exhibited

higher levels of mortality relative to controls that had

undergone adult eclosion (Fig. 8), but persisted for lon-

ger than expected.

actinLhSpot

actinLhSpot

actinLhSpot

actinLhSpot

E 1 2 3 4 0 24 48 72 0 24 48

H T A

H B G RT

H B G RT

NT

NT

NT

NT

instar 5th instar (h) adult (h)

5th instar

adult male

adult female

A

B

Figure 5. Reverse Transcription-PCR (RT-PCR) analysis of Lygus

hesperus Spookiest (LhSpot) expression. (A) Developmental expression

profile. LhSpot and actin (control gene) transcripts were amplified from

eggs (E), first to fourth instars (1–4), fifth instars at four time intervals

postmoult and adults at three intervals postmoult. (B) Tissue expression

profile in fifth instars and adults of each gender. LhSpot and actin

transcripts were amplified from head (H), thorax (T) and abdomen (A) in

fifth instars, and head, body (B), midgut/hindgut (G) and reproductive

tissue (RT) in mature adults. Female reproductive tissues include ovary

and seminal depository; male reproductive tissues include testes and

lateral/medial accessory glands. Amplimers correspond to �500-bp frag-

ments of the transcripts of interest. PCR products were electrophoresed in

1.5% agarose gels and stained with SYBR Safe. For better clarity, nega-

tive images of representative gel images are shown. NT, no template.

Image shown is representative of three biological replicates.



Effects of exogenous 20E and 7-dehydrocholesterol

following LhSpot knockdown

As Spot catalyses a relatively early step in 20E biosyn-

thesis (presumably within the so-called ‘black box’), we

hypothesized that application of ecdysteroids (eg 20E)

upstream of that step should be able to rescue the

knockdown phenotype as demonstrated in other sys-

tems (Ono et al., 2006; Jia et al., 2015; Shahzad et al.,

2015). Topical application of 20E 24 h post-LhSpot

dsRNA injection rescued adult eclosion (Fig. 9A). Non-

treated and ethanol (20E vehicle) treated nymphs failed

to enter the final moult. The rescue effect appeared to

be highly time-dependent, as addition of 20E at the time

of injection or later than 24 h post-injection either failed

to rescue adult eclosion or resulted in partial eclosion

with the adults trapped in the old exoskeleton (Fig. 9B).

buffer

EGFP dsRNA

LhSpot dsRNAin

ject

ions

actin Spot E74 E75 GAPDH

non-injected LhSpot dsRNA

3 days post-injection >20 days post-injection

non-injected LhSpot dsRNA

5 mm

0.5 mm

A

B

C non-injected LhSpot dsRNA

6-day-old adult 25-day-old nymph

Figure 6. RNA interference-mediated knockdown of Lygus hesperus Spookiest (LhSpot). (A) LhSpot transcript expression is reduced following LhSpot

double-stranded RNA (dsRNA) injection. Reverse Transcription-PCR (RT-PCR) amplification of the control genes actin and glyceraldehyde 3-phosphate

dehydrogenase (GAPDH) along with two ecdysone responsive genes (E74 and E75) and LhSpot in non-injected, enhanced green fluorescent protein

(EGFP) dsRNA-injected and LhSpot dsRNA-injected nymphs at 48 h post-injection. Amplimers correspond to �500-bp fragments of the transcripts of inter-

est except for GAPDH, which was full length. PCR products were electrophoresed in 1% agarose gels and stained with SYBR Safe. For better clarity, nega-

tive images of representative gel images are shown. Image shown is representative of three biological replicates. (B) LhSpot knockdown prevents adult

eclosion. Non-injected newly emerged fifth instars and nymphs injected with LhSpot dsRNA were imaged 3 days post-injection and> 20 days post-injection.

Non-injected controls underwent adult eclosion, whereas LhSpot dsRNA-injected nymphs retained a nymphal phenotype but continued to grow and devel-

oped abdominal pigmentation. (C) Oogenesis in LhSpot dsRNA-injected nymphs. Representative images of ovaries from a 6-day-old untreated adult female

and a nymph> 20 days after injection with LhSpot dsRNA.



We next assessed the effects of topically applied

7-dehydrocholesterol, a substrate upstream of the

ecdysteroid biosynthesis ‘black box’. Unlike 20E, exoge-

nous 7-dehydrocholesterol failed to restore adult eclo-

sion (Fig. 9A), suggesting that LhSpot catalyses an

enzymatic step downstream of 7-dehydrocholesterol.

Discussion

Despite extensive molecular and biochemical characteri-

zation of the ecdysteroid biosynthetic pathway in holo-

metabolous insects, our understanding of this pathway

in hemimetabolous pests, in particular mirids, is

extremely limited. To address this deficiency, we sought

to mine recent Ly. hesperus transcriptome assemblies

for potential orthologues of the CYP307 family (Spo,

Spok and Spot) of P450 monooxygenases, the proposed

rate-limiting enzymes in the 20E biosynthetic pathway.

The lone orthologue identified from the search aligned

more closely with Spot orthologues than with Spo or

Spok orthologues. Consistent with other Spo-like

sequences, LhSpot has a membrane-targeting hydro-

phobic amino terminus followed by a cluster of Arg/Lys

residues and a Pro/Gly-rich region that function as a

halt-transfer signal and a molecular hinge, respectively,

and motifs (the heme-binding loop, helices C, I and K,

and PERF domain) characteristic of cytochrome P450s

(Werck-Reichhart & Feyereisen, 2000). Close inspection

of insect CYP307 sequences reveals a clear differentia-

tion in the region identified as helix C between Spo/

Spok and Spot proteins. In Spo/Spok, this region is

composed of His/Tyr-Cys-Ser/Gly-Pro, whereas Spot

proteins have substituted Thr/Ile-Phe/Glu for the Cys-

Ser/Gly pair (Fig. 2). Additional sequence divergence is

seen �20 amino acids upstream of this region in which

the conserved Leu in the Ala-Leu-Cys-Asp motif in Spo/

Spok proteins is substituted with Phe in Spot proteins.

Similar paralogue variation is seen on either side of helix

I. On the amino terminal side, the conserved Phe in the

Leu-Glu-Asp-Phe sequence in Spo/Spok is substituted

with Ile in Spot. On the carboxyl side of helix I, Spot pro-

teins have substituted a charged basic amino acid (Lys)

for the hydrophobic residue (Met/Leu) in Spo/Spok

(Fig. 2). Although the impact of these amino acid substi-

tutions on the enzymatic activity or substrate specificity

of the CYP307 monooxygenases is unknown, we sug-

gest that identification of these sites may facilitate future

discrimination and annotation of Spot paralogues in

other species.

LhSpot is expressed throughout Ly. hesperus develop-

ment, with transcripts amplified from eggs, all nymphal

stages and reproductively mature adults (Fig. 5A). The

lone CYP307 paralogue in Ap. mellifera, Spot, is likewise

expressed in multiple life stages (pupa, prepupa and

non-injected

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250

2

4

6

8

10

12

14

16

18

days post-injection

Num

ber o

f ins

ects adult

nymph

buffer inject

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250

2

4

6

8

10

12

14

16

18

days post-injection

Num

ber o

f ins

ects

EGFP dsRNA inject

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250

2

4

6

8

10

12

14

16

18

days post-injection

Num

ber o

f ins

ects

LhSpot dsRNA inject

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250

2

4

6

8

10

12

14

16

18

days post-injection

Num

ber o

f ins

ects

Figure 7. Lygus hesperus Spookiest (LhSpot) double-stranded RNA-

injected fifth instars do not undergo normal adult development. The devel-

opmental status of newly emerged fifth instars was tracked for 25 days

after treatment. Data represent mean 6 SEM of samples ran in triplicate

with 15 insects per treatment. Abbreviation: EGFP, enhanced green fluo-

rescent protein.



queen ovary; Yamazaki et al., 2011). This contrasts with

the expression profile reported for the Spot orthologue

in T. castaneum, which was spatially and temporally

restricted to the adult male tubular accessory glands

(Hentze et al., 2013). No amplimer was detected in Ly.

hesperus accessory glands in our RT-PCR based analy-

ses. This difference may be attributable to very low tran-

script levels in the accessory glands, which would raise

questions regarding functional relevance, or could indi-

cate a temporal aspect to LhSpot expression in this tis-

sue. Alternatively, because two CYP307 paralogues

(Spo and Spot) were reported in T. castaneum, with Spo

expression limited to pre-adult stages, it is possible that

the two gene products fulfil specific functions that have

diverged temporally and spatially. Expression of two

paralogues in Dr. melanogaster is likewise defined in

terms of tissue and developmental stage. The Dr. mela-

nogaster Spo is expressed during early embryonic

development and adult ovarian maturation, whereas

activity of Spok is restricted to the prothoracic gland in

late embryos and larval stages (Ono et al., 2006).

Rewitz et al. (2007) speculated that the distinct spatial

and temporal expression profiles exhibited by the Spo

paralogues reflect integration of a retrosequence near

the promoter region, a genomic event that could alter

the tissue expression specificity. Gene duplication result-

ing in multiple Spo paralogues may also have facilitated

the development of distinct roles in ecdysteroid develop-

ment as evidenced by the restricted expression of Spot

in T. castaneum. The tissue-specific expression profile

exhibited when multiple Spo paralogues are present

could be an indication that LhSpot, which appears to

lack temporal and spatial restrictions on expression, is

the lone Spo paralogue in Ly. hesperus.

For Ly. hesperus, circulating ecdysteroids peak �48 h

after the final nymphal–nymphal moult, with adult eclo-

sion occurring 48 h later (Brent et al., 2016). The ele-

vated expression of LhSpot at 24 h and subsequent

decline (Fig. 5A) correlate well with the biosynthesis of

ecdysteroids needed to trigger adult eclosion. LhSpot

transcript expression, however, exhibits rhythmicity

within the fifth-instar stage as evidenced by the expres-

sion peaks at 24 and 72 h post-emergence. Similar fluc-

tuations in the expression of Halloween genes have

been reported in La. striatellus (Jia et al., 2013a, 2014,

2015; Wan et al., 2014a,b), Spodoptera littoralis (Iga &

Smagghe, 2010), Ch. suppressalis (Shahzad et al.,

2015), Bo. mori (Ono et al., 2006), Manduca sexta (Ono

et al., 2006) and T. castaneum (Hentze et al., 2013). We

speculate that the second burst of LhSpot expression

might be linked with ecdysteroid influence on the pacing

of gonadal development, which may be a response to

the loss of negative feedback associated with disintegra-

tion of the nymphal prothoracic gland (Marchal et al.,

2010), or may signify a non-ecdysteroidal function of

LhSpot. To examine the validity of these possible explan-

ations, however, further studies are needed to develop a

more thorough understanding of the physiological and

endocrinological mechanisms underlying this process in

Ly. hesperus.

RNAi-mediated knockdown of LhSpot transcripts in

newly eclosed fifth instars was apparent within 24 h of

dsRNA injection with phenotypic effects that extended

throughout normal adult eclosion, which occurred 3–4

days post-injection in control nymphs (Fig. 7). Surpris-

ingly, the LhSpot knockdown group never developed

external adult features; rather they retained the nymphal

form until death, which in some cases was 37 days after

fifth instar emergence. Delayed development was

reported following RNAi knockdown of Spo in T. casta-

neum, which had a 40% moult rate 9 days post-dsRNA

treatment (Hentze et al., 2013), in So. furcifera with 24%

remaining as third instars (Jia et al., 2013b) and Ch.

suppressalis with �25% remaining as fourth instars

(Shahzad et al., 2015). Similarly, RNAi knockdown of the

larval Spok paralogue in Drosophila resulted in �98% of

the larvae remaining as first instars (Ono et al., 2006).

By contrast, Spo knockdown in Schistocerca gregaria

and An. gambiae had no clear developmental phenotype

despite reduced ecdysteroid production (Marchal et al.,

2011; Pondeville et al., 2013). The incomplete develop-

ment that we observed compared with the delayed

development reported in other studies could be attribut-

able to methodological variations in dsRNA delivery or

biological compensatory effects. For both possibilities,

residual enzyme activities resulting from partial or tran-

sient knockdown may be sufficient to sustain ecdyste-

roids at levels necessary to allow continued

development. Indeed, Sc. gregaria Spo transcript levels

in male nymphs rebounded 2 days post-injection

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250

20

40

60

80

100S

urvi

val %

days post-injection

blank injectno inject

EGFP injectLhSpot inject

Figure 8. Lygus hesperus Spookiest (LhSpot) double-stranded RNA-

injected fifth instar females exhibit reduced survivorship. The survivorship

of newly emerged fifth instars was tracked for 25 days after treatment.

Data represent mean 6 SEM of samples ran in triplicate with 15 insects

per treatment replicate. Abbreviation: EGFP, enhanced green fluorescent

protein.



(Marchal et al., 2011). As ecdysteroidogenesis can be

regulated by multiple factors (Marchal et al., 2010), it is

possible that some regulatory feedback mechanism may

compensate for the transcript knockdown triggered by

the dsRNAs.

The rescue effect associated with exogenous applica-

tion of an ecdysteroid (eg 20E) upstream of impaired

Halloween gene activity has been demonstrated in

diverse species including So. furcifera, Dr. melanogaster

and Ch. suppressalis (Ono et al., 2006; Jia et al.,

2013a,b; Shahzad et al., 2015). Similarly, we found that

topical application of 20E at 24 h post-dsRNA injection

was sufficient to restore development to adulthood. How-

ever, no rescue effect was seen when 20E was applied

later than 24 h after injection, suggesting that proper

timing of the ecdysone pulse, perhaps in relation to juve-

nile hormone titres, is critical for normal development to

adulthood in Ly. hesperus. Consequently, it is possible

the addition of exogenous 20E later than 24 h post-

injection disrupts the timing, duration and/or interaction

framework of the ecdysteroid pulse critical to perpetuate

development to completion. Although our understanding

of the role that ecdysteroids have in hemimetabolous

insects continues to develop, it is clear that 20E affects

LhSpot dsRNA injected

+EtOH+20E

+CHCl3+7dC

+EtOH +CHCl3

LhSpot dsRNA + EtOH/20E

5 mm

B

A

non-injected0

20

40

60

80

100

% o

f ins

ects

(5

day

s po

st-tr

eatm

ent)

(n=39) (n=16) (n=37) (n=52) (n=20) (n=37)

complete moult partial moult

no adult adult moult

moult

Figure 9. Lygus hesperus Spookiest (LhSpot) knockdown effect on adult eclosion is rescued with exogenous 20-hydroxyecdysone (20E). (A) Newly

emerged fifth instars injected with LhSpot double-stranded RNA (dsRNA) were treated 24 h after injection with handling stimulus alone or topical applications

of 90% ethanol (1EtOH), 20E in 90% ethanol (120E), 100% chloroform (1CHCl3) and 7-dehydrocholesterol in 100% chloroform (17dC). The developmental

status was then tracked over a 5-day period and compared with non-injected nymphs. Under normal conditions, fifth instars typically undergo adult eclosion

after 4–5 days at 278C. The number of surviving individuals assessed at day 5 is indicated above the bars. (B) LhSpot knockdown nymphs enter eclosion fol-

lowing exogenous 20E application. Timing of the 20E application is critical to the adult moult as variations in application resulted in nymphs that only under-

went a partial moult (right insect).



an intricate transcriptional regulation cascade, disruption

of which can lead to uncoordinated development, poorly

timed moults and impaired ecdysis (Cruz et al., 2008;

Man�e-Padr�os et al., 2010, 2012).

In some female insects, ovarian-based ecdysteroido-

genesis is required for oogenesis (Terashima et al.,

2005; Parthasarathy et al., 2010), with Halloween gene

expression reported in female reproductive tissues of Dr.

melanogaster (Ono et al., 2006), Sc. gregaria (Marchal

et al., 2011), Holcocerus hippophaecolus (Zhou et al.,

2013), An. gambiae (Pondeville et al., 2013) and T. cas-

taneum (Parthasarathy et al., 2010). RNAi-mediated

knockdown of Spo and Phantom in An. gambiae and T.

castaneum, respectively, impeded ovarian ecdysteroid

production (Pondeville et al., 2013), whereas in T. casta-

neum knockdown of Shade, the terminal enzyme in the

ecdysteroid biosynthetic pathway, severely impaired ovar-

ian growth and primary oocyte maturation (Parthasarathy

et al., 2010). Similarly, Dr. melanogaster females with a

mutation in Spo failed to produce progeny when mated

with fertile wild-type males and had arrested egg devel-

opment (Ono et al., 2006). The expression of LhSpot in

adult female reproductive tissues (Fig. 5B) is consistent

with the role described above for Halloween genes in

ovarian maturation. Given the necessity of ecdysteroid

production for egg development, it was surprising to

observe oogenesis comparable to a reproductive 3-day-

old adult female in LhSpot dsRNA-injected nymphs that

had failed to undergo adult eclosion (Fig. 6C). The exter-

nal characteristics of these nymphs were phenotypically

fifth instar in appearance without wings or a protruding

ovipositor. We speculate that the internal development of

adult characteristics (ie oogenesis) is dependent on

ecdysteroid biosynthesis and was able to proceed

because the knockdown effects had diminished. Alterna-

tively, because RNAi-induced knockdown does not com-

pletely eliminate the target protein, residual enzyme

activities may be sufficient to sustain a level of 20E syn-

thesis above the threshold needed for oogenesis. These

findings also suggest that the transcriptional timing of the

two ecdysteroid-linked events, adult eclosion and oogen-

esis, is disconnected. The exact role of LhSpot in oogen-

esis and the degree of linkage between the ecdysteroid

events remain to be fully explored.

This is, to our knowledge, the first in vivo demonstra-

tion of Spot function in insect development. Here, we

have shown that the LhSpot sequence aligns phyloge-

netically with CYP307B1 proteins, that it is expressed

throughout Ly. hesperus development, and is expressed

in female reproductive tissues. The phenotypic effects

observed in immatures (uncoordinated development,

arrested adult eclosion) following RNAi knockdown,

expression throughout larval and adult development, and

singularity in our transcriptome assembly, suggest that,

as in Ap. mellifera, the LhSpot paralogue is the only

CYP307 gene in Ly. hesperus. The presence of multiple

Spo/Spok/Spot paralogues in the genomes of two hemi-

pteran pests, Ac. pisum and Ni. lugens, suggests that

loss of a former gene duplication may be a relatively

recent evolutionary event.

Experimental procedures

Insects

Lygus hesperus were reared in a laboratory colony at the

United States Department of Agriculture (USDA) Agricultural

Research Service, Arid Land Agricultural Research Center

(Maricopa, AZ, USA) and maintained on a mix of green bean

pods (Phaseolus vulgaris L.) and an ad libitum artificial diet mix

(Debolt, 1982) packaged in Parafilm M (Pechiney Plastic Pack-

aging, Chicago, IL, USA) as described previously (Patana,

1982). Insects were reared under a light : dark 14:10 h photo-

period at 27 6 1 8C and 40–60% relative humidity (RH). Eggs

were deposited in oviposition packets (Parafilm M filled with

agarose gel). Daily monitoring of experimental insects ensured

timely collection of individuals within 24 h of nymphal moult or

adult eclosion.

Transcriptomic identification and bioinformatic

characterization of a Lygus Spo-like sequence

To identify putative Ly. hesperus Spo paralogues, we used the

tBLASTn program to search (e-value�1025) our previously

assembled Ly. hesperus transcriptome (Hull et al., 2014) using

queries consisting of Spo sequences from six insect species: Dr.

melanogaster (NP_647975), Bo. mori (NP_001104833), Na. vitri-

pennis (XP_001603435), T. castaneum (XP_974280), Ac. pisum

(XP_001945761) and La. striatellus (AFU86444). The longest iso-

form from the resulting Ly. hesperus sequence hit was then re-

evaluated by BLASTx (e value�1025) using the National Center

for Biotechnology Information (NCBI) non-redundant (nr) database.

To determine phylogenetic relationships, a MUSCLE-based

multiple sequence alignment (Edgar, 2004) consisting of the puta-

tive Ly. hesperus Spo-like (LhSpot) sequence and protein sequen-

ces for the suite of Halloween genes from five species (Table S1)

was performed using default settings in GENEIOUS 7.1.8 (Biomat-

ters Ltd., Auckland, New Zealand). Phylogenetic trees were con-

structed using the maximum likelihood, minimum evolution,

UPGMA and neighbour-joining methods in MEGA6 v. 6.06

r6140220 (Tamura et al., 2013). A more detailed analysis of the

potential phylogenetic relationships between the putative LhSpot

and the sequences that comprise the CYP307A1/CYP307A2/

CYP307B1 (ie Spo/Spok/Spot) group of enzymes was performed

using 20 sequences annotated accordingly in the NCBI database.

The evolutionary history was inferred as before in MEGA6 using

the maximum likelihood method. Initial tree(s) for the heuristic

search were obtained by applying the neighbour-joining method

to a matrix of pairwise distances estimated using a Jones Thorn-

ton-Taylor (JTT) model. The tree was drawn to scale, with branch

lengths measured in the number of substitutions per site. The

analysis involved 21 amino acid sequences, with all positions con-

taining gaps and missing data eliminated, resulting in a final data

set consisting of 58 total positions. WoLF PSORT (Horton et al.,



2007) and the Target P 1.1 server (Emanuelsson et al., 2007)

were used for subcellular localization prediction.

Cloning and transcriptional profiling of LhSpot

To generate a full-length clone of LhSpot, total RNA was iso-

lated from five mixed gender fifth-instar nymphs pooled at 2

days post-eclosion (period when circulating ecdysteroid levels

are highest; Brent et al., 2016) using TRI Reagent (Life Tech-

nologies, Carlsbad, CA, USA) as described by Chomczynski &

Sacchi (1987). First-strand cDNAs were generated using Super-

script III reverse transcriptase (Life Technologies) with custom-

made random pentadecamers (IDT, San Diego, CA, USA) and

500 ng DNase I-treated total RNAs. Full-length LhSpot was

amplified using primers (Table S2) designed to span the pre-

dicted ORF of the putative LhSpot identified in our transcrip-

tome database search. Multiple independent reactions were

performed using Premix ExTaq (Clontech Laboratories, Moun-

tain View, CA, USA) in a 20-ll volume with 1 ll cDNA template

(25 ng) and 0.2 lM of each primer. Thermocycler conditions

consisted of 95 8C for 2 min followed by 35 cycles at 94 8C for

20 s, 55 8C for 20 s, 72 8C for 90 s, and a final extension at

72 8C for 5 min. Amplimers were separated on 1.5% agarose

gels using a Tris/acetate/ethylenediaminetetraacetic acid buffer

system and visualized with SYBR Safe (Life Technologies).

Products from each reaction were subcloned using a pCR2.1-

TOPO TA cloning kit (Life Technologies) and sequenced at the

Arizona State University DNA Core Laboratory (Tempe, AZ,

USA).

To examine the expression profile of LhSpot, total RNAs were

isolated as above from pooled samples of eggs, each of the first

four nymphal instars, as well as fifth instars at 0, 1, 2 and 3 days

post-ecdysis, and mixed gender adults sampled at 0, 1 and 2

days post-eclosion. For first and second instars, 20 mixed-gender

nymphs were used. For third-fifth instars, 10 mixed-gender

nymphs were pooled. For adult samples, five of each gender

were pooled. Parallel analyses examining the tissue distribution

of the LhSpot transcript used total RNAs isolated from mixed-

gender fifth instars and adults of each gender: 15 heads, five

thoraces and five abdomens for fifth instars, 25 mixed hindgut/

midgut for adults, five pairs of pooled lateral and medial acces-

sory glands, five pairs of testes and pooled samples of five pairs

of ovaries and 20 seminal depositories. Expression profiles for

each sample were generated using Sapphire Amp Fast PCR

Master Mix (Clontech Laboratories), 12.5 ng of each respective

cDNA template, and 0.2 lM of primers (Table S2) designed to

amplify nt 1–555 of a reference gene, Lygus actin (DQ386914),

or a fragment of LhSpot (nt 1–498). Thermocycler conditions con-

sisted of 95 8C for 2 min followed by 35 cycles at 94 8C for 20 s,

56 8C for 20 s and 72 8C for 30 s, and a final extension at 72 8C for

5 min. PCR products were separated on 1.5% agarose gels as

before with representative amplimers subcloned into pCR2.1-

TOPO and the sequences verified.

Transient expression of a fluorescent LhSpot chimera

in cultured insect cells

To examine the intracellular localization of LhSpot, insect expres-

sion vectors encoding fluorescent chimeras of LhSpot and an

endoplasmic reticulum marker (KDEL receptor 1) were con-

structed. Overlap extension PCR (Wurch et al., 1998) using

KOD Hot Start DNA polymerase (Toyobo/Novagen, EMD Bio-

sciences, San Diego, CA, USA) was used to generate the

respective chimeras with mVenus or mCherry fused in-frame to

the carboxyl terminal residues of LhSpot (LhSpot-Venus) and the

Homo sapiens KDEL endoplasmic reticulum protein retention

receptor 1 (HsKDELR-mCherry), respectively. The initial PCR

products were generated from plasmid DNA templates

(pCR2.1TOPO/LhSpot, above; pOTB7/HsKDELR, NM_006801.2;

Transomic Technologies, Huntsville, AL, USA; and pIB vectors

containing either mVenus or mCherry) using gene-specific and

chimeric primers (Table S2). Thermocycler conditions consisted

of 95 8C for 2 min followed by 21 cycles at 95 8C for 20 s, 58 8C

for 20 s and 70 8C for 60 s, with a final incubation at 70 8C for 5

min. Amplimers of the expected sizes were gel excised and puri-

fied using an EZNA Gel Extraction kit (Omega Bio-Tek Inc., Nor-

cross, GA, USA). The respective 5’ and 3’ fragments were

joined using KOD Hot Start DNA polymerase with gene-specific

primers (Table S2). Thermocycler conditions consisted of 95 8C

for 2 min followed by 25 cycles at 95 8C for 20 s, 56 8C for 20 s,

and 70 8C for 90 s with a final incubation at 70 8C for 5 min. The

resulting PCR products were gel excised, treated with ExTaq

DNA polymerase (Clontech) to add 3’A overhangs, cloned into

the pIB/V5-His TOPO TA insect expression vector (Life Technol-

ogies) and the sequences verified.

Trichoplusia ni cells (Orbigen Inc., San Diego, CA, USA),

maintained as adherent cultures in serum-free insect culture

media (Orbigen Inc.), were seeded into 35-mm #1.5 glass bottom

dishes (MatTek Corp., Ashland, MA, USA) and allowed to settle

for 20 min. Cells were then co-transfected with 2 lg of each plas-

mid (pIB/LhSpot-Venus and pIB/HsKDELR-mCherry) using 8 ll

Insect Gene Juice transfection reagent (Novagen, EMD Bioscien-

ces, San Diego, CA, USA) for 5 h. Transfection media was then

removed, the cells washed twice with 1 ml serum-free media and

then maintained in serum-free media at 28 8C. After 48 h, the

transfected cells were washed twice with 1 ml IPL-41 insect

media (Life Technologies) and then imaged in 2 ml IPL-41 using

a 603 phase contrast water-immersion objective (numerical

aperture 1.2) on a Fluoview FV10i-LIV laser scanning confocal

microscope (Olympus, Center Valley, PA, USA). Images were

subsequently processed (tone and contrast) in ADOBE PHOTOSHOP

CS6 (Adobe System Inc., San Jose, CA, USA).

dsRNA synthesis and injection

dsRNA was produced using a MEGAscript RNAi kit (Life Tech-

nologies) according to the manufacturer’s instructions. A 547-bp

fragment corresponding to nt 846–1352 of the LhSpot ORF was

amplified from plasmid DNA using Sapphire Amp Fast PCR

Master Mix with primers containing a 5’ T7 promoter sequence

(Table S2). The amplified fragment was TOPO-cloned into

pCR2.1-TOPO (Life Technologies) and chemically competent

TOP10 Escherichia coli cells (Life Technologies) were trans-

formed. As a negative control, a dsRNA targeting the complete

ORF of EGFP was generated using similar primers (Table S2).

The template in the T7 reaction was amplified from the plasmid

DNA with Sapphire Amp Fast PCR Master Mix and gel purified

using the EZNA gel extraction kit. After transcription, the



dsRNAs were digested with nuclease to remove residual tem-

plate DNA and non-annealed single-stranded RNA and then

purified according to the manufacturer’s instructions on columns

provided with the MEGAscript RNAi kit. The dsRNA fragments

were quantitated using the Take3 multi-volume plate on a Syn-

ergy H4 hybrid multi-mode microplate reader (BioTek Instru-

ments, Winooski, VT, USA) and then diluted to a concentration

of 1 mg/ml in MEGAscript RNAi kit elution buffer (EB).

Injection needles were made from 5-ml disposable soda lime

glass pipettes (Thermo Fisher, Kimble Chase, Pittsburgh, PA,

USA) using a Narishige PN-30 Magnetic Glass Microelectrode

Horizontal Puller (Narishige International, Amityville, NY, USA)

at heater level 84.4 and magnet level 25.5. To facilitate injec-

tion, sharp edges were made by snapping off the needle tips.

The manual dsRNA delivery system consisted of a 60-cm-long

tygon tube with a 1000-ml pipette tip inserted into one end (nar-

row tip protruding for clasping the pulled glass needles) and a

second 1000-ml pipette tip inserted in the opposite orientation at

the other end of the tube. The glass needles were fastened in

the narrow opening with Parafilm M. To calibrate the needles,

0.25 ml (for nymphs) or 0.5 ml (for adults) of RNase-free water

pipetted with a 2.5-ml pipettor was drawn directly from the pip-

ette tip using the manual injection system. The needles were

marked and the calibration was repeated three times to ensure

accuracy. Before injection, the insects were immobilized by

cooling at 4 8C for 20 min. A freezer icepack, cleaned with etha-

nol, was used as the injection stage. Insects were injected on

the ventral right side between the fifth and seventh abdominal

tergites, then allowed to recover for 10 min. Those failing to

recuperate were discarded. Survivors were maintained at 27 8C

and 30% RH in Huhtamaki waxed cups (Huhtamaki, De Soto,

KS, USA) with a mesh screen, and provided green bean pods

(Ph. vulgaris L.) every 2 days.

Fifth instars were collected within 24 h of the nymphal moult.

Three biological replicates were conducted, each having four

treatment groups: non-injected controls; nymphs injected with

0.25 ml EB; nymphs injected with 250 ng/0.25 ml EGFP dsRNA;

and nymphs injected with 250 ng/0.25 ml LhSpot dsRNA. Each

treatment and replicate consisted of 15 nymphs, which were

monitored daily for the incidence and timing of adult eclosion.

After a minimum of 25 days post-injection, survivors were dis-

sected and phenotype images collected using a Leica DFC425

camera attached to a Leica M165C microscope with an LED

ring light (Leica Microsystems, Buffalo Grove, IL, USA).

RT-PCR verification of RNAi-mediated LhSpot

knockdown

Total RNA was isolated from fifth instars (three per treatment)

as described above. Potentially contaminating genomic DNA

was removed using a DNA-free DNA removal kit (Life Technolo-

gies) according to the manufacturer’s instructions. First-strand

cDNA was synthesized from 2 mg DNase-free RNA with a RET-

ROscript kit (Life Technologies) in a 20-ml reaction according to

the manufacturer’s instructions using the supplied oligo-(dT)

primer. Full-length Ly. hesperus GAPDH (nt 1–1002; Transcrip-

tome Shotgun Assembly (TSA) accession GBHO01012854)

and �500-bp fragments of Lygus actin (nt 1–555), the ecdy-

sone responsive genes E74 (nt 61–568; TSA accession

GBHO01008524) and E75 (nt 1–556; TSA accession

GBHO01011368), and LhSpot (nt 1–498) were PCR amplified

using Sapphire Amp Fast PCR Master Mix in a 20-ml reaction

containing 0.5 ml cDNA template (50 ng) and 0.2 mM of each

primer (Table S2). Thermocycler conditions consisted of initial

denaturation at 95 8C for 2 min followed by 35 cycles at 95 8C

for 20 s, 56 8C for 20 s, and 72 8C for 60 s with a final extension

at 72 8C for 5 min. PCR products were electrophoresed as

before but using 1% agarose gels containing SYBR Safe.

Amplimers were visualized using an ImageReader LAS4000

(Fujifilm, Tokyo, Japan), subcloned as before and the sequen-

ces validated.

Exogenous application of 20E and 7-dehydrocholesterol

In total, 280 fifth-instar females over three experimental repli-

cates were collected within 24 h of moulting and injected with

250 ng/0.25 ml LhSpot dsRNA. Injected nymphs were placed in

groups of 10 within Petri dishes (10 3 1.5 cm) for overnight

maintenance. At 24 h post-injection, the insects were collected

together in a single vial and dead nymphs removed. The insects

were immobilized by cooling at 4 8C for 20 min and then treated

with 0.25 ml (1 mg/ml) 20E (Sigma-Aldrich, St Louis, MO, USA)

in 90% ethanol, 0.25 ml 90% ethanol alone, 0.25 ml (1 mg/ml)

7-dehydrocholesterol (Sigma-Aldrich) in 100% chloroform, or

0.25 ml 100% chloroform alone. One Petri dish was used for

every 10 treated nymphs and the nymphs were allowed to

recover for 2 h, after which dead nymphs were removed. A non-

replicated group of 30 non-injected female nymphs, subject only

to handling stress, was also monitored for adult eclosion under

the same conditions (10 insects/Petri dish). Petri dishes with

insects were maintained at 27 8C and 30% RH and provided

with a green bean pod section that was replaced every 2 days.

Insects were surveyed daily to determine timing of the adult

moult.

Acknowledgements

The authors thank Daniel Langhorst for maintaining the

Ly. hesperus colony and Lynn Forlow Jech for assis-

tance with tissue dissections and insect cell culture

maintenance. Funding was provided by the China Schol-

arship Council to M.W. and by Cotton Inc. (12-373) to

C.S.B. and J.J.H. Mention of trade names or commercial

products in this article is solely for the purpose of provid-

ing specific information and does not imply recommen-

dation or endorsement by the U. S. Department of

Agriculture. USDA is an equal opportunity provider and

employer.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article at the publisher’s web-site:

Table S1. MUSCLE-based multiple sequence alignment heat map of the per

cent amino acid identities amongst select insect spook (Spo)/spookier

(Spok)/Spookiest (Spot) sequences.

Table S2. List of oligonucleotide primers.

Table S3. Accession/model numbers for Halloween sequences used in

phylogenetic analyses.



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