SU PPLEMENTARY INFORMATION - media.nature.com · The migratory brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae), is the most serious insect pest of rice, a major

Supplementary note 1. Introduction of planthoppers

The migratory brown planthopper (BPH), Nilaparvata lugens (Hemiptera:

Delphacidae), is the most serious insect pest of rice, a major food source for more

than 50% of the world’s population1. The BPH is monophagous and feeds by inserting

its stylets into the vascular tissue of the rice leaf sheaths and ingesting phloem sap.

Dense BPH infestations can cause complete wilting and drying of rice plants, referred

to as hopperburn2,3. The BPH also transmits plant pathogens including rice ragged

stunt virus (RRSV; genus Oryzavirus) and rice grassy stunt virus (RGSV; genus

Tenuivirus)4,5. In the past half century, BPHs outbreaks have re-occurred

approximately every three years, with the annual outbreak area amounting to

approximately 10-20 million hectares of rice, resulting in millions of tons of losses in

Asia6,7.

In addition of BPH, Laodelphax striatellus planthopper (LSP) and Sogatella

furcifera planthopper (SFP) are another two economically important insect pests

causing yield losses on paddy fields in eastern Asia7. Analogous to BPH, LSP and SFP

damage rice mechanically by directly piercing-sucking and egg-laying, but also act as

disease vectors, transmitting various plant pathogens such as rice stripe virus (RSV;

genus Tenuivirus), rice black streaked dwarf virus (RBSDV; genus Fujivirus), and

southern rice black-streaked dwarf virus (SRBSDV; genus Fijivirus)5,8,9. The three

species are adapted to different temperatures, and differ in the geographic areas where

they overwinter. The northern geographic limit of BPH and SFP winter breeding is

approximately 21-25°N10, whereas LSP can overwinter locally as diapausing nymphs

on wheat or various weeds. Although the three species posses migratory ability, BPH

and SFP mainly damage rice in the tropics to about 42-44°N, whereas LSP mainly

infests rice in temperate areas.

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Supplementary note 2. Wing dimorphism in BPHs

One evolutionary adaptation that is thought to be essential to the success of BPH is its

striking wing dimorphism. Both female and male BPHs can develop into either a

long-winged (LW) morph or a short-winged (SW) adult morph in response to

environmental cues. Both morphs are morphologically indistinguishable during the

nymphal stages. The capability to develop into the LW morph enables BPHs to

migrate over long distances, resulting in extensive damage to rice production across

wide geographic areas. During the spring and summer, LW BPHs migrate northward

from tropical or subtropical areas as rice becomes available in temperate areas of

China, northern India, Japan and Korea11. In the autumn, returning migrations (from

north to south) of BPH populations have been observed across China and India12,13.

Most adults in subsequent post-migration generations are SW morphs and exhibit

increased fecundity14. Although numerous studies suggest that wing dimorphism is

associated with various environmental cues, such as population density, rice nutrient

conditions, genetic variation, and even developmental hormones15-20, the exact

regulatory mechanism controlling wing dimorphism in the BPH remains largely

unknown.

Supplementary note 3. Sequence analysis of NlInR1 and NlInR2

In contrast to the single insulin receptor (InR) found in the Diptera (flies and

mosquito), at least two InRs have been identified in some Hemiptera, Hymenoptera,

Lepidoptera, Coleoptera, and Isoptera. The two InRs of BPH are called NlInR1 and

NlInR2. To identify the structural differences between NlInR1 and NlInR2 that might


SUPPLEMENTARY INFORMATIONRESEARCHdoi:10.1038/nature14286

define their distinct physiological roles, we determined the primary structures of

NlInRs from cloned cDNAs. The full-length cDNA sequences of NlInR1 and NlInR2

consist of 5,835 bp (Extended Data Fig. 1a) and 6,004 bp (Extended Data Fig. 1b)

excluding poly(A) tails, which are predicted to encode 1,454 and 1,427 amino acid

receptor precursors, respectively. The predicted mass of NlInR1 and NlInR2 are 158.4

and 158.9 kDa, respectively, excluding the signal peptide. NlInR1 and NlInR2 closely

resemble their Drosophila and human orthologs with respect to domain architecture

(Extended Data Fig. 1c). In the extracellular region of NlInR1 and NlInR2, two

ligand-binding loops (L1 and L2) with one furin-like cysteine rich region (Fu)

between them were identified (Extended Data Fig. 1a,b). There are three fibronectin

type 3 (Fn3) domain repeats in both NlInRs, which are important for the formation of

two disulphide bonds when the α- and β-subunits dimerize. A single transmembrane

domain (TM) is predicted downstream of the third Fn3 region in both NlInRs. In the

intracellular region, a juxtamembrane NPXY motif resides (Extended Data Fig. 1a,b,

indicated in green) in NlInR1 and NlInR2, which is important for optimal

phosphorylation of the insulin receptor substrate (IRS-1) 21,22. Following NPXY motif,

a conversed tyrosine kinase domain contains a regulatory region YXXXYY motif, of

which autophosphorylation of the three tyrosine residues likely fully stimulates the

kinase activity23.

A noticeable difference between NlInR1 and NlInR2 is that the latter lacks four

cysteine residues (Extended Data Fig. 1d) in the amino-terminal part of Fu domain

that plays an important functional role in the interaction of the receptor with

insulin24,25. The four cysteine residues which are absent in NlInR2 occupy conserved



positions in the corresponding regions of the NlInR1, Drosophila and human insulin

receptors. Additionally, NlInR2 appears to contain more post-translational

modification sites than NlInR1. We predicted nineteen potential N-linked

glycosylation sites in NlInR2, as opposed to eleven sites in NlInR1. Furthermore, ten

potential tyrosine phosphorylation sites are predicted in the cytoplasmic region of

NlInR2, compared to six sites in NlInR1. These distinct structural features may

contribute to the physiologically different effects of NlInR1 and NlInR2 in the brown

planthopper.

To better understand the functional differences of NlInR1 and NlInR2, we

investigated their spatio-temporal expression patterns. The results indicated that

NlInR1 was widely expressed at all stages and in all of the tissue examined (Extended

Data Fig. 2a-c, g). By contrast, NlInR2 was enriched in 4th- and 5th-instar nymphs,

with a particularly strong expression in the wing buds (Extended Data Fig. 2d,e,g),

consistent with its role in wing development.

Supplementary note 4. Intermediate forewing length in planthoppers with

NlInR2 and NlAkt knockdown

We observed both severe (Fig. 2c) and moderate (Fig. 2d) RNAi phenotypes in

individuals treated with a dsRNA mixture of dsNlInR2 and dsNlAkt. The moderate

phenotype, consisting of an intermediate forewing length (Fig. 2d, arrowhead), was

observed in 29.5% of females (n=88) and 65% of males (n=112). This intermediate

phenotype suggests that dsNlAkt failed to completely neutralize the dsNlInR2 effect

when 4th-instar nymphs were used for RNAi. Here, we refer to BPHs with an

intermediate forewing length as SW BPHs because they also have undeveloped

hindwings.

Supplementary note 5. Tissue-specific regulation by NlInR2



We asked whether the long-winged morph generated by dsNlInR2 was due to the

tissue specific regulation via NlInR2 or was an indirect consequence of the effect on

growth. We found (1) no discernible difference of hind tibiae length between SW

BPHs (dsgfp-SW) and LW BPHs (dsgfp-LW), indicating that wing morph switch is

independent of body growth. BPHs treated either with dsNlInR2 or with double-gene

RNAi (dsNlInR2;dsNlFoxo) have hind tibiae of similar lengths to those of dsgfp-LW

(Extended Data Fig. 6a), indicating that NlInR2 specifically regulates the growth of

wing buds rather than being involved in the systemic regulation of appendage tissues;

(2) BPHs treated with dsNlInR2 or dsNlInR2;dsNlFoxo possess forewings of similar

size (Extended Data Fig. 6b), although slightly smaller than long-winged BPHs

(dsgfp-LW); (3) knockdown of NlInR1, NlChico or NlAkt further reduced the hind

tibiae length (Extended Data Fig. 6c) and forewing size (Extended Data Fig. 6d)

compared to SW BPHs treated with dsgfp (dsgfp-SW). Regardless of the decrease in

size, the wing veins were positioned almost normally in dsRNAs-treated SW BPHs

(Fig. 2b); (4) knockdown of NlInR1 or NlChico but not NlInR2 severely delayed

nymphal development (Extended Data Fig. 6e); (5) knockdown of NlInR1 but not

NlInR2 in 2nd-instar nymphs resulted in body weight loss in 5th-instar nymphs

(Extended Data Fig. 6f); (6) knockdown of NlInR1 but not NlInR2 reduced

whole-body glycogen, trehalose, and glucose contents in nymphs (Extended Data Fig.

6g-i) as well as in adult females (Extended Data Fig. 6j-l). Interestingly, these

observations stand in contrast to findings in Drosophila, which showed increased

levels of these molecules when IIS activity was compromised, for instance in Chico

mutants26, in InR mutants27, and in flies with ablation of cells making insulin-like

ligands28. The underlying mechanism of the species-dependent discrepancy remains

elusive; (7) knockdown of NlInR1 but not NlInR2 increased whole-body triglyceride



content both in nymphs (Extended Data Fig. 6m) and adult females (Extended Data

Fig. 6n). Since perturbation of the insulin signaling pathway in Drosophila severely

impairs organismal growth, body size and life span, in addition to metabolism26-31, our

findings show that NlInR1, but not NlInR2, stimulates this well-established insulin

response pathway in the BPH. NlInR2, by contrast, regulates wing morph

development in a tissue-specific way rather than through a systemic effect on growth.

Supplementary note 6. Subcellular localization of NlFOXO in wing buds

We investigated the NlFOXO subcellular localization in wing buds and fat body

following treatments of dsNlInR2, dsNlPten, dsNlInR1, dsgfp, and PI3K inhibitor

LY294002. We found that dsNlInR2 excluded the NlFOXO from the nucleus in wing

buds (Extended Data Fig. 7a,c) but not in the fat body (Extended Data Fig. 7b,c), in

contrast to the cytoplasmic accumulation of NlFOXO in both tissues treated with

dsNlPten. By contrast, LY294002 treatment resulted in nuclear accumulation of

NlFOXO in both tissues (Extended Data Fig. 7a, b). However, we did not observe

biased subcellular localization of NlFOXO in the wing buds and fat body treated with

either dsgfp or dsNlInR1. The latter might be due to the fact that the IIS activity was

not completely eliminated by NlInR1 RNAi silencing. To confirm these observations,

we investigated the P-NlAkt level in fat body treated with various dsRNAs. We found

that knockdown of NlPten but not NlInR2 increase P-NlAkt levels (Extended Data Fig.

7d), which was in contrary to elevated P-NlAkt levels in wing buds treated with either

dsNlPten or dsNlInR2 (Fig.2f). In conclusion, these data suggest that NlInR2

determines alternative wing morphs in a tissue-specific way through regulating the

downstream factor NlFOXO.

Supplementary note 7. Tissue distribution of NlILP3 in the BPH

To gain additional information on the function of NlILP3, we investigated the tissue



distributions of each insulin peptide. The results revealed that each NlIlp gene showed

its own distinctive spatial expression pattern (Extended Data Fig. 8). Notably, NlIlp3

was observed to be predominantly expressed in the head in 4th- and 5th-instar nymphs,

and in adult females (Extended Data Fig. 8g-i). An immunofluorescence assay with

His-NlILP3 antiserum revealed that NlILP3 was specifically expressed in two clusters

of medial neurosecretory cells (MNCs) located alongside the medial furrow and in

some bilateral endocrine cells (NCs) in the brains of 5th-instar nymphs (Fig. 3c, and

Supplementary Video 1). Collectively, the above data indicate that the brain-secreted

NlILP3 is the main insulin peptide that initiates NlInR1-NlPI3K-NlAkt signaling to

promote long wing development in BPHs.

Supplementary note 8. A common mechanism in planthopper family

We employed an additional two planthopper species, S. furcifera (SFP) (Extended

Data Fig. 9a) and L. striatellus (LSP) (Extended Data Fig. 9c), to investigate whether

the two insulin receptors played common roles in wing morph switching. We found

that dsSfInR1 treatment dramatically increased the ratio of SW morphs in SFP

(Extended Data Fig. 9b). In the parallel experiment, LW morphs were observed

following dsSfInR2 treatment (Extended Data Fig. 9b), as expected. Given that SFP

tends to develop into the long-winged morph under laboratory rearing conditions, the

observed effect of dsSfInR1 treatment offers more convincing evidence of the

regulatory mechanism of the insulin/IGF signaling pathway.

By contrast, L. striatellus planthopper (LSP) (Extended Data Fig. 9c) species

serves as an excellent model for investigating the effect of dsLsInR2 treatment

because this species tends to develop into the short-winged morph under laboratory

conditions. We observed that dsLsInR2 treatment robustly reduced the proportion of

SW morphs in SFP (Extended Data Fig. 9d). However, dsLsInR1 treatment only



marginally increased the proportion of SW males in LSP (Extended Data Fig. 9d). It

is worth noting that most of dsLsInR1-treated nymphs died before metamorphosis

although no significant change on survival rate was observed at beginning nine days

(Extended Data Fig. 9e). In the following days, less than 30% of dsLsInR1-treated

nymphs, in contrary to about 80% of dsgfp-treated nymphs, could molt into adults

(Extended Data Fig. 9f). We speculated that the intrinsic InR1 level in LSP is relative

low compared to that found in BPH and SFP. This hypothesis is supported by the

finding that nearly all short-winged LSPs (but few short-winged SFPs) were produced

when both species were raised in the same chamber. Thus, further reducing InR1 level

by RNAi in LSF might severely impair the ability of nymphs to reach the critical

weight for metamorphosis.

Supplementary note 9. Full scan for figures2f-i





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SU PPLEMENTARY INFORMATION - media.nature.com · The migratory brown planthopper (BPH), Nilaparvata lugens (Hemiptera: Delphacidae), is the most serious insect pest of rice, a major