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Citation: Ahmed, J.; Walker, A.A.;
Perdomo, H.D.; Guo, S.; Nixon, S.A.;
Vetter, I.; Okoh, H.I.; Shehu, D.M.;
Shuaibu, M.N.; Ndams, I.S.; et al.
Two Novel Mosquitocidal Peptides
Isolated from the Venom of the Bahia
Scarlet Tarantula (Lasiodora klugi).
Toxins 2023,15, 418. https://doi.org/
10.3390/toxins15070418
Received: 18 May 2023
Revised: 16 June 2023
Accepted: 23 June 2023
Published: 27 June 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
toxins
Article
Two Novel Mosquitocidal Peptides Isolated from the Venom
of the Bahia Scarlet Tarantula (Lasiodora klugi)
Jamila Ahmed 1,2, Andrew A. Walker 2,3 , Hugo D. Perdomo 4, Shaodong Guo 2,3 , Samantha A. Nixon 2,3 ,
Irina Vetter 2,5 , Hilary I. Okoh 6, Dalhatu M. Shehu 1, Mohammed N. Shuaibu 7,8, Iliya S. Ndams 1,
Glenn F. King 2,3 and Volker Herzig 2,9,10,*
1Department of Zoology, Ahmadu Bello University Zaria, Kaduna 810107, Nigeria
2Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
3
Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, University
of Queensland, Brisbane, QLD 4072, Australia
4School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia
5School of Pharmacy, The University of Queensland, Brisbane, QLD 4102, Australia
6Department of Animal and Environmental Biology, Federal University Oye-Ekiti, Oye 371104, Nigeria
7Department of Biochemistry, Ahmadu Bello University Zaria, Kaduna 810107, Nigeria
8Centre for Biotechnology Research and Training, Ahmadu Bello University Zaria, Kaduna 810107, Nigeria
9Centre for Bioinnovation, University of the Sunshine Coast, Sippy Downs, QLD 4556, Australia
10 School of Science, Technology, and Engineering, University of the Sunshine Coast,
Sippy Downs, QLD 4556, Australia
*Correspondence: vherzig@usc.edu.au
Abstract:
Effective control of diseases transmitted by Aedes aegypti is primarily achieved through
vector control by chemical insecticides. However, the emergence of insecticide resistance in
A. aegypti
undermines current control efforts. Arachnid venoms are rich in toxins with activity against dipteran
insects and we therefore employed a panel of 41 spider and 9 scorpion venoms to screen for mosquito-
cidal toxins. Using an assay-guided fractionation approach, we isolated two peptides from the
venom of the tarantula Lasiodora klugi with activity against adult A. aegypti. The isolated peptides
were named U-TRTX-Lk1a and U-TRTX-Lk2a and comprised 41 and 49 residues with monoisotopic
masses of 4687.02 Da and 5718.88 Da, respectively. U-TRTX-Lk1a exhibited an LD
50
of 38.3 pmol/g
when injected into A. aegypti and its modeled structure conformed to the inhibitor cystine knot
motif. U-TRTX-Lk2a has an LD
50
of 45.4 pmol/g against adult A. aegypti and its predicted structure
conforms to the disulfide-directed
β
-hairpin motif. These spider-venom peptides represent potential
leads for the development of novel control agents for A. aegypti.
Keywords:
Lasiodora klugi; insecticidal toxin; Aedes aegypti; disulfide-directed
β
-hairpin; inhibitor
cystine knot
Key Contribution:
We discovered two novel peptides from the venom of the tarantula Lasiodora klugi
that might be used to control Aedes aegypti mosquitoes and limit spread of the diseases they transmit.
1. Introduction
Arboviral diseases like dengue, yellow fever, chikungunya, and zika are primarily
vectored by the mosquito Aedes aegypti [
1
,
2
]. Dengue is the most important arboviral
disease transmitted by A. aegypti, with an estimated 390 million annual cases and over
half of the world population in 129 countries at risk of infection [
2
,
3
]. Yellow fever, an
acute hemorrhagic viral disease, accounts for over 29,000 annual deaths with the disease
being endemic in 47 countries [
1
]. Chikungunya is a mosquito-borne viral disease that
has been identified in 42 countries in Africa, Asia, the Americas, and Europe [
4
]. Zika
virus infections have been reported in Asia, Africa, the Pacific, and the Americas, with its
infection being associated with microcephaly in Brazil [5].
Toxins 2023,15, 418. https://doi.org/10.3390/toxins15070418 https://www.mdpi.com/journal/toxins
Toxins 2023,15, 418 2 of 14
Successful control of these diseases is mainly achieved through vector control using
chemical insecticides. However, the emergence of resistance in A. aegypti to major insecticide
classes such as pyrethroids, carbamates, and organophosphates threatens the control
of these diseases [
6
,
7
]. Furthermore, chemical insecticides affect non-target beneficial
organisms, cause environmental pollution, and threaten human health upon inhalation or
accidental consumption. A safe alternative could be insect-selective toxins that have been
patented for their possible use to control insect vectors [8].
Spiders are among the most successful and diverse venomous animals with an esti-
mated 120,000 extant species [
9
], of which ~51,000 belonging to 132 families have been
formally recognized [
10
]. Having evolved during the Ordovician period (450 million
years ago), they are found in all types of habitats except for polar regions, the highest
mountains, and the oceans [
8
]. Their success is partially attributed to the use of venom to
rapidly subdue prey which mainly consists of a wide variety of other arthropods, including
medically important disease vectors and agricultural pests [
11
–
13
]. Thus, spiders play an
important ecological role in keeping insect populations at bay [
14
]. To enable spiders to
overcome diverse types of prey, their venom comprises a complex mixture of active biologi-
cal components, including proteins, peptides, acylpolyamines, small amines, histamine,
and other small molecules [
12
,
15
–
17
]. Peptides are the most abundant components in their
venom, with cysteine-rich peptides being the most important functional component [
17
].
Several insecticidal peptides have been isolated from the venom of various spider species
and some have been patented as bio-insecticidal leads. One of these peptides has been
developed commercially by Vestaron Corporation [
8
]. According to the ArachnoServer
spider toxin database, over 230 spider-venom peptides have been reported to be insectici-
dal based on experimental data or predictions based on sequence homology [
18
]. These
peptides act on a diverse range of molecular targets, including voltage-gated calcium chan-
nels, voltage-gated sodium channels, calcium-activated potassium channels, presynaptic
nerve terminals, lipid bilayers, nicotinic acetylcholine receptors, and N-methyl-D-aspartate
(NMDA) receptors [8,19–21].
Scorpions are the oldest group of arachnids, having evolved in the Silurian pe-
riod
[22,23]
. To date, 2722 species have been described [
24
], and they are widely distributed
and adapted to different terrestrial habitats except for Antarctica. Like spiders, their success
is attributed to their use of venom for prey capture and defense against predators [
25
].
Their venoms are a complex mixture of bioactive compounds such as inorganic salts, amino
acids, nucleic acids, peptides, mucopolysaccharides, and proteins [
26
,
27
]. Therefore, spider
and scorpion venoms are considered rich repositories for the discovery of novel toxins to
control insect pest and vectors [8,20,21,28–31].
Efficient delivery is considered the primary obstacle to deployment of venom-derived
peptides as bio-control agents [
32
]. In 2007, the entomopathogenic fungus Metarhizium
anisopliae was engineered to express AaIT, a toxin isolated from the venom of the scor-
pion Androctonus australis. The transgenic fungus was observed to reduce the kill time
of A. aegypti as compared to the wild-type fungus [
33
]. Similarly, a pathogenic fungus
(Metarhizium pingshaense) engineered with an insect-specific toxin from spider venom was
reported to reduce the Anopheles mosquito population during a field trial in Burkina Faso
by 90% [
34
]. Furthermore, in 2017 the US Environmental Protection Agency approved use
of the SPEAR
™
(Vestaron, Durham, NC, USA) range of bioinsecticides, in which the active
component is a peptide derived from the venom of an Australian funnel-web spider [
35
].
To further expand the insecticidal armory against disease vectors such as mosquitoes,
our study was designed to screen a diverse panel of 50 arachnid venoms with the aim
of isolating and characterizing venom peptides that are active against adult A. aegypti
mosquitoes.
Toxins 2023,15, 418 3 of 14
2. Results
2.1. Isolation and Purification of Mosquitocidal Tarantula Venom Peptides
We performed a preliminary insecticidal screen of 41 spider and 9 scorpion
venoms by injecting 6.25 ng of each venom into five adult A. aegypti mosquitoes
(
Supplementary Tables S1 and S2
). Potent mortality of >50% was observed in 19 ven-
oms (=38%) at 24 h post-injection with 12 tarantula venoms causing 80–100% mortality
(
Supplementary Figure S1
), while none of the scorpion venoms caused mortality above
20% (
Supplementary Table S2
). The venom of L. klugi caused 100% irreversible paralysis
at 0.5 h post injection leading to over 90% mortality after 2 h. Bioassay-guided fraction-
ation of L. klugi venom using a C
18
reversed-phase (RP) HPLC column revealed that the
fractions eluting from 26 to 28 min (Figure 1A) caused irreversible contractile paralysis
in 100% of injected mosquitoes and ultimately death after 24 h. These fractions were
sub-fractionated using a hydrophilic interaction liquid chromatography (HILIC) column
(Figure 1B,C), which resulted in the isolation of two active peptides which eluted at ~12 min
and 15 min, and were named U-TRTX-Lk1a (Lk1a) and U-TRTX-Lk2a (Lk2a) according to
the rational nomenclature for peptide toxins [
36
]. Mass spectrometric analysis revealed the
monoisotopic mass to be 4687.02 Da for Lk1a and 5718.88 Da for Lk2a (Figure 1D,E).
Toxins 2023, 15, x FOR PEER REVIEW 3 of 14
study was designed to screen a diverse panel of 50 arachnid venoms with the aim of
isolating and characterizing venom peptides that are active against adult A. aegypti
mosquitoes.
2. Results
2.1. Isolation and Purification of Mosquitocidal Tarantula Venom Peptides
We performed a preliminary insecticidal screen of 41 spider and 9 scorpion venoms
by injecting 6.25 ng of each venom into five adult A. aegypti mosquitoes (Supplementary
Tables S1 and S2). Potent mortality of >50% was observed in 19 venoms (=38%) at 24 h
post-injection with 12 tarantula venoms causing 80–100% mortality (Supplementary
Figure S1), while none of the scorpion venoms caused mortality above 20%
(Supplementary Table S2). The venom of L. klugi caused 100% irreversible paralysis at 0.5
h post injection leading to over 90% mortality after 2 h. Bioassay-guided fractionation of
L. klugi venom using a C18 reversed-phase (RP) HPLC column revealed that the fractions
eluting from 26 to 28 min (Figure 1A) caused irreversible contractile paralysis in 100% of
injected mosquitoes and ultimately death after 24 h. These fractions were sub-fractionated
using a hydrophilic interaction liquid chromatography (HILIC) column (Figure 1B,C),
which resulted in the isolation of two active peptides which eluted at ~12 min and 15 min,
and were named U-TRTX-Lk1a (Lk1a) and U-TRTX-Lk2a (Lk2a) according to the rational
nomenclature for peptide toxins [36]. Mass spectrometric analysis revealed the
monoisotopic mass to be 4687.02 Da for Lk1a and 5718.88 Da for Lk2a (Figure 1D,E).
1430 1431 1432 1433 1434
0
100
200
300
400
500
600
700
m/z
Intensity
1430.72 Da
0 102030
0
50
100
150
0
20
40
60
80
100
Time (min)
Absorbance (214 nm)
% of solvent B
U2-TRTX-LK1a
0 102030405060
0
500
1000
1500
2000
3000
4000
5000
0
20
40
60
80
100
Time (min)
Absorbance (214 nm)
% of solvent B
f28
f29
1563 1564 1565 1566
0
250
500
750
1000
1250
m/z
Intensity
1563.34 Da
0 102030
0
100
200
300
0
20
40
60
80
100
Time (min)
Absorbance (214 nm)
% of solvent B
U1-TRTX-LK1a
A
BC
DE
U
1
-TRTX-Lk1a CGGVDAPCDKDRPDCCSYAECLKPAGYGWWHGTYYCYRKKE
U
2
-TRTX-Lk1a FFECTFECDIKKEGKPCKPKGCKCKDKDNKDHKKCSGGWRCKLKLCLKF
F
Figure 1. Isolation and purification of mosquitocidal peptides.
(
A
) The crude venom of female
Lasiodora klugi (pictured in the insert, photo courtesy of Bastian Rast) was fractionated using RP-HPLC
and fractions 28 and 29 exhibited mosquitocidal activity against A. aegypti. (
B
,
C
) Further fractionation
using HILIC-HPLC resulted in the purification of U-TRTX-Lk1a from fraction 28 and U-TRTX-Lk2a
from fraction 29 (red arrows pointing to respective toxin peaks). (
D
,
E
) The molecular masses (black
arrows pointing to the monoisotopic masses) were identified by electrospray mass spectrometry
as 4687.02 Da for U-TRTX-Lk1a (based on the 3
+
ion) and 5718.88 Da for U-TRTX-Lk2a (based on
the 4
+
ion). (
F
) Peptide sequences for U-TRTX-Lk1a and U-TRTX-Lk2a as determined by Edman
degradation and de novo sequencing. Cysteine residues are highlighted in red.
Toxins 2023,15, 418 4 of 14
2.2. Mosquitocidal ACTIVITY
Upon injection into A. aegypti, both toxins caused irreversible contractile paralysis
resulting in 100% mortality after 24 h. Lk1a had an LD
50
of 38.3 pmol/g (Figure 2A), while
Lk2a had an LD50 of 45.4 pmol/g after 24 h (Figure 2B).
Toxins 2023, 15, x FOR PEER REVIEW 4 of 14
Figure 1. Isolation and purification of mosquitocidal peptides. (A) The crude venom of female
Lasiodora klugi (pictured in the insert, photo courtesy of Bastian Rast) was fractionated using RP-
HPLC and fractions 28 and 29 exhibited mosquitocidal activity against A. aegypti. (B,C) Further
fractionation using HILIC-HPLC resulted in the purification of U-TRTX-Lk1a from fraction 28 and
U-TRTX-Lk2a from fraction 29 (red arrows pointing to respective toxin peaks). (D,E) The molecular
masses (black arrows pointing to the monoisotopic masses) were identified by electrospray mass
spectrometry as 4687.02 Da for U-TRTX-Lk1a (based on the 3+ ion) and 5718.88 Da for U-TRTX-Lk2a
(based on the 4+ ion). (F) Peptide sequences for U-TRTX-Lk1a and U-TRTX-Lk2a as determined by
Edman degradation and de novo sequencing. Cysteine residues are highlighted in red.
2.2. Mosquitocidal ACTIVITY
Upon injection into A. aegypti, both toxins caused irreversible contractile paralysis
resulting in 100% mortality after 24 h. Lk1a had an LD50 of 38.3 pmol/g (Figure 2A), while
Lk2a had an LD50 of 45.4 pmol/g after 24 h (Figure 2B).
Figure 2. Mosquitocidal activity. Dose–response curves for the toxicity of the venom peptides Lk1a
(A) and Lk2a (B) as observed 24 h after injection into adult Aedes aegypti.
2.3. Primary Structure Determination for Mosquitocidal Venom Peptides
Using a combination of Edman sequencing and de novo liquid chromatography–
tandem mass spectrometry (LC-MS/MS), complete peptide sequences were determined
for both Lk1a and Lk2a. For Lk1a, Edman sequencing at the Australian Proteome Analysis
Facility returned the partial N-terminal sequence
CGGVDAPCDKKRPDCCS(S)AECLK(P)AG-(G), with brackets indicating a tentative
assignment. This sequence closely matches the previously reported peptide U2-TRTX-
Lsp1a CGGVDAPCDKDRPDCCSSAECLKPAGYGWWHGTYYCYRKRER from Lasiodora
sp. (hps://arachnoserver.qfab.org/toxincard.html?id=509, accessed 27 March 2022).
Manual analysis of LC-MS/MS data from reduced, alkylated, and trypsinized Lk1a
U-TRTX-Lk2a
10
-12
10
-11
10
-10
10
-9
0
20
40
60
80
100
Dose (mol/g)
% Dead
LD
50
= 45.4 ± 3.8 pmol/g
U-TRTX-Lk1a
10
-12
10
-11
10
-10
10
-9
0
20
40
60
80
100
Dose (mol/g)
% Dead
LD
50
= 38.3 ± 5.9 pmol/g
B
A
Figure 2. Mosquitocidal activity.
Dose–response curves for the toxicity of the venom peptides Lk1a
(A) and Lk2a (B) as observed 24 h after injection into adult Aedes aegypti.
2.3. Primary Structure Determination for Mosquitocidal Venom Peptides
Using a combination of Edman sequencing and de novo liquid chromatography–
tandem mass spectrometry (LC-MS/MS), complete peptide sequences were determined for
both Lk1a and Lk2a. For Lk1a, Edman sequencing at the Australian Proteome Analysis Fa-
cility returned the partial N-terminal sequence CGGVDAPCDKKRPDCCS(S)AECLK(P)AG-
(G), with brackets indicating a tentative assignment. This sequence closely matches the
previously reported peptide U
2
-TRTX-Lsp1a CGGVDAPCDKDRPDCCSSAECLKPAGYG-
WWHGTYYCYRKRER from Lasiodora sp. (https://arachnoserver.qfab.org/toxincard.html?
id=509, accessed 27 March 2022). Manual analysis of LC-MS/MS data from reduced, alky-
lated, and trypsinized Lk1a revealed that the isolated peptide ends in TYYCYRKKE (car-
boxyl terminus), indicating a conservative R41K polymorphism compared to the database
peptide, with the final R residue likely removed by carboxypeptidase similar to other spider-
venom peptides [
12
]. Examination of matrix-assisted laser desorption/ionization (MALDI-
TOF) MS spectra with fragmentation induced by 1,5-diaminonaphthalene (
1,5-DAN
) yielded
the internal fragmentary sequence DCCSYAE, indicating Y rather than the S that was ten-
tatively called by Edman sequencing at the 18th residue. Taken together, the combined
data yields a putative sequence of CGGVDAPCDKDRPDCCSYAECLKPAGYGWWHG-
Toxins 2023,15, 418 5 of 14
TYYCYRKKE with a predicted monoisotopic mass of 4686.973 Da, which closely matched
the measured monoisotopic mass of this peptide (4687.02 Da).
For Lk2a, manual de novo peptide sequencing from LC-MS/MS data yielded the tryp-
tic fragment CSGGWR. A search of the ArachnoServer database [
18
] with this fragment re-
turned the peptide U
1
-TRTX-Lsp1a FFECTFECDIKKEGKPCKPKGCKCKDKDNKDHKKC-
SGGWRCKLKLCLKF from Lasiodora sp. venom (https://arachnoserver.qfab.org/toxincard.
html?id=669, accessed 27 March 2022) with a predicted monoisotopic mass of 5718.80 Da,
which closely matches the measured monoisotopic mass of the native toxin, 5718.88 Da.
To further examine the mass spectral evidence for these peptide primary structures,
we compared the LC-MS/MS data of reduced, alkylated, and trypsinized peptides with
a sequence database containing the putative Lk1a and Lk2a sequences, all amino acid
sequences from Arachnida on UniProt, and 200 common MS contaminants. Lk1a and
Lk2a were the top detected peptides in each sample. Three tryptic peptides originating
from Lk1a were confidently detected, covering 49% of the sequence. Five tryptic peptides
originating from Lk2a were detected, covering 53% of the sequence, including the 20 N-
terminal residues of the peptide. Together, the MS data provides good support for the
determined primary structures of these peptides (Figure 3).
Toxins 2023, 15, x FOR PEER REVIEW 5 of 14
revealed that the isolated peptide ends in TYYCYRKKE (carboxyl terminus), indicating a
conservative R41K polymorphism compared to the database peptide, with the final R
residue likely removed by carboxypeptidase similar to other spider-venom peptides [12].
Examination of matrix-assisted laser desorption/ionization (MALDI-TOF) MS spectra
with fragmentation induced by 1,5-diaminonaphthalene (1,5-DAN) yielded the internal
fragmentary sequence DCCSYAE, indicating Y rather than the S that was tentatively called
by Edman sequencing at the 18th residue. Taken together, the combined data yields a
putative sequence of CGGVDAPCDKDRPDCCSYAECLKPAGYGWWHGTYYCYRKKE
with a predicted monoisotopic mass of 4686.973 Da, which closely matched the measured
monoisotopic mass of this peptide (4687.02 Da).
For Lk2a, manual de novo peptide sequencing from LC-MS/MS data yielded the
tryptic fragment CSGGWR. A search of the ArachnoServer database [18] with this
fragment returned the peptide U1-TRTX-Lsp1a
FFECTFECDIKKEGKPCKPKGCKCKDKDNKDHKKCSGGWRCKLKLCLKF from
Lasiodora sp. venom (hps://arachnoserver.qfab.org/toxincard.html?id=669, accessed 27
March 2022) with a predicted monoisotopic mass of 5718.80 Da, which closely matches
the measured monoisotopic mass of the native toxin, 5718.88 Da.
To further examine the mass spectral evidence for these peptide primary structures,
we compared the LC-MS/MS data of reduced, alkylated, and trypsinized peptides with a
sequence database containing the putative Lk1a and Lk2a sequences, all amino acid
sequences from Arachnida on UniProt, and 200 common MS contaminants. Lk1a and Lk2a
were the top detected peptides in each sample. Three tryptic peptides originating from
Lk1a were confidently detected, covering 49% of the sequence. Five tryptic peptides
originating from Lk2a were detected, covering 53% of the sequence, including the 20 N-
terminal residues of the peptide. Together, the MS data provides good support for the
determined primary structures of these peptides (Figure 3).
Figure 3. Primary structure determination. The primary structure of Lk1 and Lk2 was determined
using a combination of N-terminal Edman sequencing, LC-MS/MS analysis, 1,5-
diaminonaphthalene induced fragmentation, and homology searches in public databases. All
residues matching with the complete sequence are indicated in bold, tentative calls from Edman are
underlined, the colors indicate different confidence levels for the de novo LC-MS/MS sequencing
(blue > 95% confidence; purple < 50% confidence). Residue numbers are shown above the sequences.
Basic local alignment searches of amino acid sequences of isolated peptides revealed
several peptides from tarantula venoms with sequence identities of 51–100%. There are 16
and 21 conserved sites in the multiple sequence alignments of Lk1a and Lk2a, respectively
1 10 2 0 30 4 0
Lk1 (complete sequence) CGGVDAPCDKDRPDCCSYAECLKPAGYGWWHGTYYCYRKKE
U2-TRTX-Lsp1a CGGVDAPCDKDRPDCCSSAECLKPAGYGWWHGTYYCYRKRER
Edman CGGVDAPCDKKRPDCCSSAECLKPAG-G
1,5 DAN DCCSYAE
LC-MS/MS (>95% confidence) CGGVDAPCDK
LC-MS/MS (>95% confidence) TYYCYRKKE
LC-MS/MS (>95% confidence) TYYCYR
1 10 20 30 40
Lk2 (complete sequence) FFECTFECDIKKEGKPCKPKGCKCDDKDNKDHKKCSGGWRCKLKLCLKF
U1-TRTX-Lsp1a FFECTFECDIKKEGKPCKPKGCKCKDKDNKDHKKCSGGWRCKLKLCLKF
LC-MS/MS (>95% confidence) FFECTFECDIKKEGKPCKPK
LC-MS/MS (>95% confidence) FFECTFECDIKKEGKPCK
LC-MS/MS (>95% confidence) EGKPCKPK
LC-MS/MS (>95% confidence) KEGKPCKPK
LC-MS/MS (>95% confidence) KEGKPCK
LC-MS/MS (>95% confidence) LKLCLK
LC-MS/MS (<50% confidence) LCLKF
LC-MS/MS (<50% confidence) CSGGWR
LC-MS/MS (<50% confidence) KCSGGWR
LC-MS/MS (<50% confidence) CKPKGCKCDDKDNK
Figure 3. Primary structure determination.
The primary structure of Lk1 and Lk2 was determined
using a combination of N-terminal Edman sequencing, LC-MS/MS analysis, 1,5-diaminonaphthalene
induced fragmentation, and homology searches in public databases. All residues matching with
the complete sequence are indicated in bold, tentative calls from Edman are underlined, the colors
indicate different confidence levels for the de novo LC-MS/MS sequencing (blue > 95% confidence;
purple < 50% confidence). Residue numbers are shown above the sequences.
Basic local alignment searches of amino acid sequences of isolated peptides revealed
several peptides from tarantula venoms with sequence identities of 51–100%. There are
16 and 21 conserved sites in the multiple sequence alignments of Lk1a and Lk2a, respec-
tively (Figure 4). All toxins with characterized molecular targets in both alignments are
modulators of voltage-gated calcium (CaV) channels.
Toxins 2023,15, 418 6 of 14
Toxins 2023, 15, x FOR PEER REVIEW 6 of 14
(Figure 4). All toxins with characterized molecular targets in both alignments are
modulators of voltage-gated calcium (Ca
V
) channels.
Figure 4. Multiple sequence alignments. The closest matching spider-venom peptide sequences for
(A) Lk1a and (B) Lk2a. Homologous sequences were identified by BLAST searches of the NCBI and
ArachnoServer) databases, and the resulting sequences were aligned using ClustalX (version 2.0).
Cysteine residues are highlighted in green and asterisks indicate conserved regions in the
alignment. The percent sequence identity to the mosquitocidal toxins from L. klugi is indicated on
the right.
2.4. In Silico Structures of Isolated Peptides
Lk1a is comprised of 41 amino acid residues, including 6 cysteines, while Lk2a is
comprised of 49 amino acids, including 8 cysteines. SWISS-MODEL
(hps://swissmodel.expasy.org, accessed 17 November 2022) and AlphaFold2 Colab
(hps://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.i
pynb, accessed 17 November 2022) was used to predict the structures of the mosquitocidal
peptides. Using SWISS-MODEL, the 30-residue peptide µ-TRTX-Pn3a (PDB code: 5T4R)
from venom of the tarantula Pamphobeteus nigricolor [37] was used as a template to model
Lk1a. Sequence alignment revealed 30% sequence identity and 0.35 global model quality
estimation (GMQE) (Figure 5A). The two peptides have a similar cysteine framework with
few conserved sites. The modeled structure conforms to the inhibitor cystine knot (ICK)
motif [38] having two antiparallel β-sheets (E20–C22 and G26–W29) connected by a loop
and a small α-helix (R12-D14) (Figure 5B). Similarly, the structure predicted by
AlphaFold2 conforms to the ICK motif (Figure 5D) having a predicted local distance
difference test of 60–85% (Figure 5C). Both modeled structures have three disulfide bonds:
C1–C16, C8–C21, and C15–C36 (Figure 5A). When the two predicted structures were
superimposed, the major difference was a string of residues connecting the two β sheets
that are longer in the structure predicted by AlphaFold2 (Figure 5E). Importantly, the
structure predicted by AphaFold2 is made up of all 41 residues, while the predicted
SWISS-MODEL structure is missing the last 10 residues (Figure 5B, D). Additionally, the
structure predicted by SWISS-MODEL has a small α helix (Figure 5B).
Figure 4. Multiple sequence alignments.
The closest matching spider-venom peptide sequences for
(
A
) Lk1a and (
B
) Lk2a. Homologous sequences were identified by BLAST searches of the NCBI and
ArachnoServer) databases, and the resulting sequences were aligned using ClustalX (version 2.0).
Cysteine residues are highlighted in green and asterisks indicate conserved regions in the alignment.
The percent sequence identity to the mosquitocidal toxins from L. klugi is indicated on the right.
2.4. In Silico Structures of Isolated Peptides
Lk1a is comprised of 41 amino acid residues, including 6 cysteines, while Lk2a is
comprised of 49 amino acids, including 8 cysteines. SWISS-MODEL (https://swissmodel.
expasy.org, accessed 17 November 2022) and AlphaFold2 Colab (https://colab.research.
google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb, accessed 17
November 2022) was used to predict the structures of the mosquitocidal peptides. Using
SWISS-MODEL, the 30-residue peptide
µ
-TRTX-Pn3a (PDB code: 5T4R) from venom of
the tarantula Pamphobeteus nigricolor [
37
] was used as a template to model Lk1a. Sequence
alignment revealed 30% sequence identity and 0.35 global model quality estimation (GMQE)
(Figure 5A). The two peptides have a similar cysteine framework with few conserved sites.
The modeled structure conforms to the inhibitor cystine knot (ICK) motif [
38
] having
two antiparallel
β
-sheets (E20–C22 and G26–W29) connected by a loop and a small
α
-
helix (R12-D14) (Figure 5B). Similarly, the structure predicted by AlphaFold2 conforms
to the ICK motif (Figure 5D) having a predicted local distance difference test of 60–85%
(Figure 5C). Both modeled structures have three disulfide bonds: C1–C16, C8–C21, and
C15–C36 (Figure 5A). When the two predicted structures were superimposed, the major
difference was a string of residues connecting the two
β
sheets that are longer in the
structure predicted by AlphaFold2 (Figure 5E). Importantly, the structure predicted by
AphaFold2 is made up of all 41 residues, while the predicted SWISS-MODEL structure
is missing the last 10 residues (Figure 5B,D). Additionally, the structure predicted by
SWISS-MODEL has a small αhelix (Figure 5B).
The structure of the Lk2a peptide was predicted using
ω
-TRTX-Br1b (PDB code:
2KGH) from Brachypelma albiceps (previously B. ruhnaui) as a template [
39
]. The peptide
and template have 82% sequence identity and 0.61 GMQE. The sequences have a similar
cysteine framework; however, Lk2a contains eight cysteines, and was predicted to have four
disulfide bonds, while
ω
-TRTX-Ba1b contains six cysteines, forming three disulfide bonds.
The sequences also differ by seven residues (indicated in yellow in Figure 6A), with a large
insertion between positions 22 and 31 in Lk2a (Figure 6A). The predicted 3D structure
of Lk2a conforms to the disulfide-directed
β
-hairpin motif [
40
] having three anti-parallel
β
-sheets stabilized by three disulfide bonds. Interestingly, this structure also contains an
α
-helix and a fourth disulfide bond. The first
β
-sheet (K15–C17) is separated from the
second
β
-sheet (G38–K41) by an
α
-helix (D32–K33) while the second
β
-sheet is connected
to the third (L45–L47) by a loop (Figure 6B). The Lk2a structure predicted by AlphaFold2
(Figure 6D) has a very low predicted local distance different test (PLDDT, Figure 6C), and
Toxins 2023,15, 418 7 of 14
as such does not give a reliable picture of the structure. None of the predicted regions
matched when both structures were overlayed (Figure 6E). Experimental analysis using
NMR spectroscopy will be required in the future to verify the predicted fold of Lk2a.
Toxins 2023, 15, x FOR PEER REVIEW 7 of 14
Figure 5. Predicted 3D structure of Lk1a. (A) Multiple sequence alignment of Lk1a with its template
µ-TRTX-Pn3a; GMQE, global model quality estimation; *, conserved site; (B) predicted structure of
Lk1a using SwissModel; (C) predicted local distance difference test per residue position using
AlphaFold2; (D) predicted structure of Lk1a using AlphaFold2; (E) overlap of SwissModel- and
AlphaFold2-predicted structures of Lk1a. The cysteine regions are highlighted in green/purple. The
N-terminal and C-terminal of all the structures are indicated in brown and red, respectively.
The structure of the Lk2a peptide was predicted using ω-TRTX-Br1b (PDB code:
2KGH) from Brachypelma albiceps (previously B. ruhnaui) as a template [39]. The peptide
and template have 82% sequence identity and 0.61 GMQE. The sequences have a similar
cysteine framework; however, Lk2a contains eight cysteines, and was predicted to have
four disulfide bonds, while ω-TRTX-Ba1b contains six cysteines, forming three disulfide
bonds. The sequences also differ by seven residues (indicated in yellow in Figure 6A), with
a large insertion between positions 22 and 31 in Lk2a (Figure 6A). The predicted 3D
structure of Lk2a conforms to the disulfide-directed β-hairpin motif [40] having three anti-
parallel β-sheets stabilized by three disulfide bonds. Interestingly, this structure also
contains an α-helix and a fourth disulfide bond. The first β-sheet (K15–C17) is separated
from the second β-sheet (G38–K41) by an α-helix (D32–K33) while the second β-sheet is
connected to the third (L45–L47) by a loop (Figure 6B). The Lk2a structure predicted by
AlphaFold2 (Figure 6D) has a very low predicted local distance different test (PLDDT,
Figure 6C), and as such does not give a reliable picture of the structure. None of the
predicted regions matched when both structures were overlayed (Figure 6E).
Experimental analysis using NMR spectroscopy will be required in the future to verify
the predicted fold of Lk2a.
Figure 5. Predicted 3D structure of Lk1a.
(
A
) Multiple sequence alignment of Lk1a with its template
µ
-TRTX-Pn3a; GMQE, global model quality estimation;
*
, conserved site; (
B
) predicted structure
of Lk1a using SwissModel; (
C
) predicted local distance difference test per residue position using
AlphaFold2; (
D
) predicted structure of Lk1a using AlphaFold2; (
E
) overlap of SwissModel- and
AlphaFold2-predicted structures of Lk1a. The cysteine regions are highlighted in green/purple. The
N-terminal and C-terminal of all the structures are indicated in brown and red, respectively.
Toxins 2023, 15, x FOR PEER REVIEW 8 of 14
Figure 6. Predicted 3D structure of Lk2a. (A) Multiple sequence alignment of Lk2a with its template
ω-TRTX-Ba1b; GMQE, global model quality estimation; *, conserved sites; ↑, insertion; ‘, mutations;
(B) predicted structure of Lk2a using SwissModel; (C) predicted local distance difference test per
residue position using AlphaFold2; (D) predicted structure of Lk2a using AlphaFold2; (E) aligned
predicted structures of Lk2a. The cysteine regions are highlighted in green/purple. The N-terminal
and C-terminal of all the structures are indicated in brown and red, respectively.
3. Discussion
Aedes aegypti is a vector of several viral diseases that have resulted in a substantial
economic burden and the loss of many human lives [1–5]. Several control measures have
been put in place to control diseases vectored by this mosquito, which are centered on
vector control achieved mainly using chemical insecticides. However, the development of
resistance to chemical insecticides by Aedes aegypti [6,7] poses a threat to this control
method. As such, there is an urgent need to develop alternative control methods. To aid
the development of eco-friendly biocontrol methods, we screened 50 arachnid venoms for
insecticidal activity against A. aegypti, and potent insecticidal activity was observed in the
venom of Bahia scarlet tarantula L. klugi. Using a combination of RP-HPLC and HILIC,
two potent insecticidal peptides, namely, Lk1a and Lk2a, were isolated from the venom
of L. klugi. These peptides caused irreversible paralysis and eventually mortality in A.
aegypti adults but differed slightly in their potency. To the best of our knowledge, these
are the first insecticidal peptides isolated by directly screening for activity against A.
aegypti mosquitoes. However, other theraphosid venom peptides with insecticidal activity
against dipterans have been reported, e.g., Ae1a causing irreversible paralysis in
Drosophila melanogaster [41] and two venom peptides from Monocentropus balfouri causing
paralysis in Lucilia cuprina and Musca domestica [42]. An important question that needs to
be addressed in future studies is how the two mosquitocidal leads Lk1a and Lk2a might
be delivered for controlling mosquitoes in the field. An approach that has already
produced promising results is the use of entomopathogens for toxin delivery [43,44].
Entomopathogens like Isaria fumosorosea have been reported to be potent against A. aegypti
[45] and could therefore be engineered to produce Lk1a and Lk2a. This method of
deployment offers several advantages like reduction in kill time, improved oral activity,
Figure 6. Predicted 3D structure of Lk2a.
(
A
) Multiple sequence alignment of Lk2a with its template
ω
-TRTX-Ba1b; GMQE, global model quality estimation;
*
, conserved sites;
↑
, insertion; ‘, mutations;
(
B
) predicted structure of Lk2a using SwissModel; (
C
) predicted local distance difference test per
residue position using AlphaFold2; (
D
) predicted structure of Lk2a using AlphaFold2; (
E
) aligned
predicted structures of Lk2a. The cysteine regions are highlighted in green/purple. The N-terminal
and C-terminal of all the structures are indicated in brown and red, respectively.
Toxins 2023,15, 418 8 of 14
3. Discussion
Aedes aegypti is a vector of several viral diseases that have resulted in a substantial
economic burden and the loss of many human lives [
1
–
5
]. Several control measures have
been put in place to control diseases vectored by this mosquito, which are centered on
vector control achieved mainly using chemical insecticides. However, the development
of resistance to chemical insecticides by Aedes aegypti [
6
,
7
] poses a threat to this control
method. As such, there is an urgent need to develop alternative control methods. To aid
the development of eco-friendly biocontrol methods, we screened 50 arachnid venoms for
insecticidal activity against A. aegypti, and potent insecticidal activity was observed in the
venom of Bahia scarlet tarantula L. klugi. Using a combination of RP-HPLC and HILIC,
two potent insecticidal peptides, namely, Lk1a and Lk2a, were isolated from the venom of
L. klugi
. These peptides caused irreversible paralysis and eventually mortality in A. aegypti
adults but differed slightly in their potency. To the best of our knowledge, these are the first
insecticidal peptides isolated by directly screening for activity against A. aegypti mosquitoes.
However, other theraphosid venom peptides with insecticidal activity against dipterans
have been reported, e.g., Ae1a causing irreversible paralysis in Drosophila melanogaster [
41
]
and two venom peptides from Monocentropus balfouri causing paralysis in Lucilia cuprina and
Musca domestica [
42
]. An important question that needs to be addressed in future studies
is how the two mosquitocidal leads Lk1a and Lk2a might be delivered for controlling
mosquitoes in the field. An approach that has already produced promising results is the
use of entomopathogens for toxin delivery [
43
,
44
]. Entomopathogens like Isaria fumosorosea
have been reported to be potent against A. aegypti [
45
] and could therefore be engineered
to produce Lk1a and Lk2a. This method of deployment offers several advantages like
reduction in kill time, improved oral activity, and increased phyletic specificity [
46
]. A
field trial in Burkina Faso using an entomopathogenic fungus engineered to express an
insecticidal spider toxin (
ω
/
κ
-hexatoxin) demonstrated that spider-venom peptides can be
successfully employed to control Anopheles populations under field conditions [34].
The sequences of the isolated mosquitocidal peptides were elucidated using Edman
degradation and LC-MS/MS analysis. Lk1a and Lk2a contain 41 and 49 residues, with a
monoisotopic mass of 4687.02 Da and 5718.88 Da, respectively (Figure 1). Both peptides
contain cysteine residues but differ in their disulfide architecture. Several anti-insect toxins
have been reported to contain cysteine residues that form disulfide bonds [
8
]. Furthermore,
a search of public databases using the Lk1a and Lk2a sequences revealed similarity with
anti-insect toxins from Brachypelma harmorii (
ω
-TRTX-Bh2a and
ω
-TRTX-Bh1a) and Brachy-
pelma albiceps (
ω
-TRTX-Ba1b). These peptides have been reported to be toxic to crickets
by inhibiting Ca
V
channels, but not toxic to mice [
39
], suggesting they might be good
insecticidal leads. Given the similarity with
ω
toxins, it seems obvious to suggest that the
two toxins isolated in this study are likely to be CaVchannel modulators.
The 3D structures of the two mosquitocidal toxins were predicted using SWISS-
MODEL and AphaFold2. Two structural motifs were predicted: the ICK, or knottin, motif
and the disulfide-directed
β
-hairpin (DDH). The ICK and DDH motifs are the most com-
mon, with ICK peptides accounting for more than 90% of toxins in some spider venoms [
42
].
The predicted Lk1a structure using AlphaFold2 Colab and SWISS-MODEL conforms with
the ICK motif (Figure 5B,D). This motif is characterized by a cystine knot [
38
] which is
potentially advantageous for insecticidal leads due to the inherently high stability of ICK
peptides [
8
,
47
–
49
]. The predicted Lk2a structure using SWISS-MODEL (Figure 6B) is an
elaborated DDH motif having an
α
-helix and four disulfide bonds. Such elaborations in
the disulfide architecture have been reported to play an important role in peptide diversi-
fication [
47
], and several studies have reported the DDH motif from spider and scorpion
venom peptides [
37
,
50
–
52
]. The Lk2a structure predicted using AlphaFold2 Colab does
not conform with any known venom peptide structure. Due to the low PLDDT (<50),
which is a measure of confidence, further experimental evidence, for example, using NMR
spectroscopy, is required to determine its 3D structure and cysteine connectivity.
Toxins 2023,15, 418 9 of 14
4. Conclusions
We discovered two mosquitocidal peptides Lk1 and Lk2 from the venom of the Bahia
scarlet tarantula Lasiodora klugi. Further experiments are required to determine the best
strategies for applying these leads under field conditions for controlling Aedes aegypti
mosquitoes and consequently the diseases they transmit.
5. Materials and Methods
5.1. Rearing of Aedes aegypti
Mosquitoes were reared according to the method of Perdomo et al. [
53
]. Plastic cups
were filled (1/3) with deionized water. Filter papers were submerged in the cups, which
were kept in cages containing adult mosquitoes. Glass plate artificial feeders using parafilm
membrane were used to blood feed female adult mosquitoes. Blood (containing acid citrate
dextrose as an anti-coagulant) was obtained from Australian Red Cross Services (Kelvin
Grove, QLD, Australia) and maintained at a temperature of 37
◦
C by circulating water
through the feeders. Following the laying of eggs on the filter papers, each filter paper
containing Aedes aegypti eggs was placed in a flat plastic tray filled with distilled water
and grounded fish feed was added. This setup was observed after 24 h for hatched larvae
and monitored every day for pupae. Pupae were transferred into fresh plastic cups which
were placed in adult cages. The emerged adults were fed 5% sucrose and maintained at
27 ±1◦C temperature, relative humidity of 75–80%, and 12 h alternating photoperiods.
5.2. Venom Extraction
Venoms were sourced from 41 spider and 9 scorpion species. Spider venoms were
collected by electrical stimulation of the basal part of the chelicerae [
54
] while scorpion
venoms were collected by forcing aggravated scorpions to sting a sheet of parafilm, from
where the venom was then collected [
13
]. After collection, the venoms were lyophilized,
and venom stock solutions were prepared by reconstituting with Milli-Q water.
5.3. Mosquito Toxicity Bioassay
For each venom, five adult female mosquitoes with an average weight of 3.19 mg
were anesthetized on ice and with the aid of a nanoinjector (Nanoject III, Drummond
Scientific Company, Broomall, PA, USA) and a binocular dissecting microscope (Nikon
SMZ800, Nikon Instrument Inc., Melville, NY, USA) and 6.25 ng of venom reconstituted in
phosphate buffer saline (PBS) was then injected into the ventrolateral thoracic region. The
mosquitoes were placed in transparent plastic cups and observed after 0.5, 1, 2, and 24 h for
paralysis or death [
13
]. This experiment was replicated thrice, and mosquitoes injected with
PBS only served as the negative control. Using a similar injection procedure, two purified
active L. klugi peptides were subjected to adulticidal toxicity assays. These consisted of
injecting 6 doses between 0.06 and 4 ng of each peptide into N = 5 mosquitoes in triplicate.
An additional N = 5 mosquitoes for each dose were injected with PBS as a control. The
resulting LD
50
was determined using a sigmoidal dose–response curve (variable slope) in
Prism 8 (Graphpad Software, San Diego, CA, USA) as previously described [13].
5.4. Peptide Isolation
Bioassay-guided fractionation was used to isolate active peptides from L. klugi venoms
by combining RP-HPLC and HILIC chromatography. For RP-HPLC, we used solvents A
(0.09% formic acid (FA) in water) and solvent B (0.09% FA in 90% acetonitrile (ACN)). The
equivalent of one milligram (based on the dried weight) of crude venom was dissolved in
5% solvent B (450
µ
L) and subjected to fractionation using a C
18
Phenomenex Jupiter RP-
HPLC column (250
×
4 mm, 5
µ
m, Phenomenex Jupiter, Sydney, Australia). Peptides were
eluted at a flow rate of 0.7 mL/min (using the following gradient: 0–5 min:
5% B
;
5–50 min
:
5–50% B
; 50–65 min: 50–100% B) and UV absorption was monitored at 214 nm [
55
]. The
resulting venom fractions were injected into mosquitoes (see Section 5.3) and the active
fractions were further subjected to HILIC-HPLC according to the method of Badgett
Toxins 2023,15, 418 10 of 14
et al. [
56
] using HILIC solvents A (trifluoroacetic acid (TFA) 0.5% in water) and B (90%
ACN in 0.043 TFA). The venom fractions were dissolved in 450
µ
L of 95% HILIC solvent
B and subfractions were eluted at a flow rate of 1 mL/min using the following gradient:
0–23 min: 95% B; 23–25 min: 75% B; 25–27 min: 5% B.
5.5. Proteomics
The purity and mass of isolated peptides were determined using MALDI-TOF mass
spectrometry on an AB Sciex TOF/TOF 5800 (Framingham, MA USA) proteomic ana-
lyzer. Toxin samples were mixed 1:1 (v:v) with
α
-cyano-4-hydroxy-cinnamic acid matrix
(6 mg/mL in 50/50 acetonitrile/H
2
O with 5% FA) and MALDI-TOF spectra were acquired
in reflector positive mode.
Similarly, 1,5-diaminonaphthalene (1,5-DAN) was used to induce fragmentation in
Lk1a by mixing it with the toxin in a 1:1 (v:v) ratio. MALDI-TOF spectra for prepared
samples were acquired using reflector positive mode.
For LC-MS/MS, native untreated peptides were prepared by diluting fractionated
peptides to 25
µ
L in 1% FA. The method of Walker et al. [
57
] was used to prepare reduced,
alkylated, and trypsinized peptides for de novo sequencing. Peptide samples (4
µ
g) were
incubated in 40
µ
L of reducing alkylating agent (4.875 mL ACN, 4.5 mL Milli-Q water,
0.5 mL 1 M ammonium carbonate pH 11.0, 100
µ
L 2-iodoethanol, 25
µ
L triethylphosphine)
for 37
◦
C for 1 h before drying. Peptides were then resuspended in 10
µ
L trypsin reagent
(40 ng/
µ
L of trypsin in 50 mM ammonium bicarbonate pH 8.0 and 10% ACN). An extraction
agent (50% ACN, 5% FA) was added to inactivate the trypsin, and prepared samples were
dried and resuspended in 40 µL of 1% FA.
Peptide samples were loaded onto a Zorbax 300SB-C18 column (Agilent #858750-
902) on a Shimadzu Nexera X2 LC system, and eluted using a 14 min gradient of 1–40%
solvent B (90% ACN/0.1% FA) in solvent A (0.1% FA) at a flow rate of 0.2 mL/min. The
LC outflow was coupled to a 5600 Triple TOF mass spectrometer (SCIEX) equipped with
a Turbo V ion source. For MS1 scans, m/zwas set between 350 and 2200. Precursor
ions with m/z350–1500, charge of +2 to +5, and signals with >100 counts/s (excluding
isotopes within 2 Da) were selected for fragmentation, and MS2 scans were collected
over a range of 80–1500 m/z. The resulting MS/MS spectra data were analyzed using
PEAKS
®
studio version 5.2 software (Bioinformatics Solutions Inc., Waterloo, ON, Canada)
for de novo sequencing. Peptide masses were calculated using online software (https:
//www.peptidesynthetics.co.uk/tools/, accessed 27 March 2020).
5.6. Edman Sequencing
The first 26 residues of Lk1a were determined using Edman degradation and the re-
maining part of the sequence was determined using de novo LC-MS/MS sequencing while
the sequence of Lk2a was determined using de novo LC-MS/MS only. N-terminal Edman
sequencing was conducted by the Australian Proteome Analysis Facility (Sydney, NSW,
Australia). Briefly, the peptide sample was solubilized in 25 mM ammonium bicarbon-
ate/10% ACN, reduced using dithiothreitol (25 mM) at 56
◦
C for 0.5 h) and then alkylated
using iodoacetamide (55 mM) at room temperature for 0.5 h. The prepared sample was then
desalted/purified by RP-HPLC using a Zorbax 300SB-C18 column (
3×150 mm
), loaded
onto a precycled, Biobrene-treated disc, and subjected to sequencing on an automated
Applied Biosystems 494 Procise Protein Sequencing System [58].
The isolated peptides were named Lk1a and Lk2a according to King et al. [
36
]. Prot-
Param (http://web.expasy.org/protparam, accessed 25 March 2020) was used to compute
parameters such as molecular weight, theoretical pI, and extinction coefficient. The result-
ing sequences were searched against a public database containing peptides from spiders
(https://arachnoserver.qfab.org/mainMenu.html, accessed 27 March 2020) and the NCBI
non-redundant (nr) database (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed 27 March
2020) using the BLASTp algorithm with the expected value (e-value) cutoff set to <10
−5
to
Toxins 2023,15, 418 11 of 14
determine similar sequences that are homologs of the insecticidal peptides [
18
,
59
]. Multiple
sequence alignments were performed with CLUSTALX v2.0.
5.7. Structure Modeling
The structures of active peptides were predicted using SWISS-MODEL) [
60
] and
AphaFold2 [
61
] Colab [
62
]. Templates were searched using target sequence with the aid of
BLAST [
63
] and HHBlits [
64
] against the SWISS-MODEL template library (SMTL). Global
model quality estimation (GMQE) was used to estimate the template-target quality. The
models were built following target-template alignment using ProMod3 and PROMOD-
II [
65
]. All templates used in this study were NMR solution structures that do not have
ligands. Peptide sequences were copied into the query input section and run on the Al-
phaFold2 Colab (https://colab.research.google.com/github/sokrypton/ColabFold/blob/
main/AlphaFold2.ipynb, accessed 17 November 2022) without a template. The resulting
structural models were downloaded and edited using PyMOL v2.4.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/toxins15070418/s1, Figure S1: Mean adulticidal activities of spider
and scorpion venoms against adult Aedes aegypti after 24 hours of observation; Table S1: Adulticidal
activity expressed in percentage of mortality of venoms isolated from spider species against adult
Aedes aegypti. Spider taxonomy according to the World Spider catalog (https://wsc.nmbe.ch/),
version 24, accessed 11 April 2023; Table S2: Adulticidal activity expressed in percentage of mortality
of venoms isolated from scorpion species against adult Aedes aegypti. Scorpion taxonomy according
to The Scorpion Files (https://www.ntnu.no/ub/scorpion-files/index.php), accessed 11 April 2023.
Author Contributions:
Conceptualization, J.A., D.M.S., M.N.S., I.S.N., V.H. and G.F.K.; Methodology,
J.A., V.H., S.G., S.A.N., H.D.P., A.A.W. and I.V. Formal Analysis, J.A., A.A.W., V.H. and H.I.O.;
Resources I.V. and G.F.K.; Data Curation, J.A., D.M.S., M.N.S., I.S.N., V.H. and A.A.W.; Validation,
V.H., G.F.K., D.M.S., M.N.S. and I.S.N.; Writing—Original Draft Preparation, J.A., V.H.; Writing—
Review and Editing, all authors; Visualization, V.H., J.A. and H.I.O.; Supervision, V.H., D.M.S., M.N.S.,
I.S.N., G.F.K.; Project Administration, V.H., D.M.S., M.N.S., I.S.N. and G.F.K.; Funding Acquisition,
J.A. and G.F.K. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the International Foundation for Science Sweden (I-1-F-6130-
1) and the Institute for Molecular Bioscience, The University of Queensland, Australia. VH was
supported by an Australian Research Council (ARC) Future Fellowship (FT190100482). AAW was
supported by the ARC through Discovery Project DP200102867; IV was supported by a National
Health and Medical Research Council (NHMRC) Investigator grant 2017086; GFK was supported
by an NHMRC Principal Research Fellowship APP1136889; and AAW, SG, SAN, and GFK were
supported as part of the ARC Centre of Excellence for Innovations in Peptide and Protein Science
CE200100012.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors thank the members of the Deutsche Arachnologische Gesellschaft
(DeArGe) for providing arachnids for venom extraction, in particular Bastian Rast, Ingo Wendt,
Michel Lüscher, and Reto Ehrler for providing L. klugi. This research was facilitated by access to
the Australian Proteome Analysis Facility supported under the Australian Government’s National
Collaborative Research Infrastructure Strategy (NCRIS).
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
Toxins 2023,15, 418 12 of 14
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