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
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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
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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
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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
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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
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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
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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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