Ci a ion: Bodláko á, K.; ˇ
Ce ný, J.;
Š ˇe bo á, H.; Gu áˇn, R.; Zí ka, O.;
Kod ík, D. Insec Body De ence
Reac ions agains Bee Venom: Do
Adipokine ic Ho mones Play a Role?.
Toxins 2022,14, 11. h ps://doi.o g/
10.3390/ oxins14010011
Recei ed: 23 No embe 2021
Accep ed: 20 Decembe 2021
Published: 23 Decembe 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
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Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
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A ibu ion (CC BY) license (h ps://
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oxins
A icle
Insec Body De ence Reac ions agains Bee Venom: Do
Adipokine ic Ho mones Play a Role?
Ka olina Bodláko á1,2,†, Jan ˇ
Ce ný1,2,† , Helena Š ˇe bo á1, Roman Gu áˇn 3,4 , Ondˇ ej Zí ka 3,4
and Dalibo Kod ík1,2,*
1Biology Cen e, Ins i u e o En omology, CAS, B anišo ská31, 370 05 Ceske Budejo ice, Czech Republic;
[email p o ec ed] (K.B.); [email p o ec ed] (J. ˇ
C.); [email p o ec ed] (H.Š.)
2Facul y o Science, Uni e si y o Sou h Bohemia, B anišo ská31a, 370 05 Ceske Budejo ice, Czech Republic
3Depa men o Chemis y and Biochemis y, Facul y o Ag iSciences, Mendel Uni e si y in B no,
Zemˇedˇelská1665/1, 613 00 B no, Czech Republic; [email p o ec ed] (R.G.); [email p o ec ed] (O.Z.)
4Cen al Eu opean Ins i u e o Technology, B no Uni e si y o Technology, Pu kyˇno a 656/123,
612 00 B no, Czech Republic
*Co espondence: [email p o ec ed]
† The au ho s con ibu ed equally o he wo k.
Abs ac :
Bees o iginally de eloped hei s inging appa a us and enom agains membe s o hei
own species om o he hi es o agains p eda o y insec s. Ne e heless, he biological and biochemi-
cal esponse o a h opods o bee enom is no well s udied. Thus, in his s udy, he physiological
esponses o a model insec species (Ame ican cock oach, Pe iplane a ame icana) o honeybee enom
we e in es iga ed. Bee enom oxins elici ed se e e s ess (LD
50
= 1.063 uL enom) esul ing in a
signi ican inc ease in adipokine ic ho mones (AKHs) in he cock oach cen al ne ous sys em and
haemolymph. Venom ea men induced a la ge des uc ion o muscle cell ul as uc u e, especially
myo ib ils and sa come es. In e es ingly, co-applica ion o enom wi h cock oach Pe am-CAH-II
AKH elimina ed his e ec . En enoma ion modula ed he le els o ca bohyd a es, lipids, and p o eins
in he haemolymph and he ac i i y o diges i e amylases, lipases, and p o eases in he midgu .
Bee enom signi ican ly educed i ellogenin le els in emales. Dopamine and glu a hione (GSH
and GSSG) insigni ican ly inc eased a e enom ea men . Howe e , dopamine le els signi ican ly
inc eased a e Pe am-CAH-II applica ion and a e co-applica ion wi h bee enom, while GSH and
GSSG le els immedia ely inc eased a e co-applica ion. The esul s sugges a gene al eac ion o he
cock oach body o bee enom and a leas a pa ial in ol emen o AKHs.
Keywo ds:
adipokine ic ho mone; Ame ican cock oach; dopamine; honey bee; meli in; me abolism;
muscle ul as uc u e; i ellogenin
Key Con ibu ion:
This s udy desc ibes he e ec o bee enom on he le els o adipokine ic ho -
mones, haemolymph nu ien s, i ellogenins, dopamine, and oxida i e s ess ma ke s; he ac i i y o
diges i e enzymes; and he muscle ul as uc u e in he body o he model species, he cock oach
(Pe iplane a ame icana). The ole o adipokine ic ho mones in physiological p ocesses a e enom
ea men is emphasised.
1. In oduc ion
Honeybee enom is a cock ail o a ious biologically ac i e subs ances including
pep ides, p o eins, amines, and amino acids dissol ed in an aqueous solu ion [
1
]. Toxins
ei he igge biochemical cascades o become biologically ac i e ollowing he e ec s o
ano he oxin h ough posi i e eedback loops. Bee enom mainly con ains meli in (ap-
p oxima ely 50% o d y ma e ), phospholipases, hyalu onidase, o he enzymes, and small,
biologically ac i e subs ances wi h ee amine g oups (dopamine, his amine, se o onin,
and no epineph ine) [
1
,
2
]. Meli in is a 26-amino acid pep ide wi h nume ous biological,
oxicological, and pha macological ac i i ies [
3
,
4
]. Toxici y is based on an abili y o bind
Toxins 2022,14, 11. h ps://doi.o g/10.3390/ oxins14010011 h ps://www.mdpi.com/jou nal/ oxins
Toxins 2022,14, 11 2 o 18
o he phospholipid bilaye o cell memb anes and o m po es. Meli in also ac i a es
o he enzymes, including phospholipases, which b eak down phospholipids and inc ease
cell des uc ion [
1
,
4
], which is he likely cause o mi ochond ial damage and collapse [
5
].
Biogenic amines also play impo an oles as majo s imula o s o in lamma ion and o he
immune esponses [
2
,
6
]. The de ence eac ion agains bee enom in he ic im’s body is
gene ally well unde s ood; howe e , no all he de ails a e known. The ecen iden i ica ion
o i ellogenin in bee enom [
7
] sugges s ha i inc eases he alle genic p ope ies o he
enom; howe e , his has no been s udied in de ail. Vi ellogenins a e mos ly known as
yolk p ecu so s in ol ed in ep oduc ion, bu hey also play impo an oles in p o ec ion
agains oxida i e s ess, wound healing, and insec immuni y, wi h s ong ac i i y agains
bac e ia and o he pa hogens [8,9].
Bee enom in oxica ion ce ainly elici s se e e s ess on he a ec ed body, wi h he an i-
s ess esponse gene ally con olled by he ne ous and endoc ine sys ems [
10
]. In insec s,
an i-s ess eac ions a e p edominan ly egula ed by adipokine ic ho mones (AKHs), which
a e esponsible o main aining homeos asis. AKHs a e small pep ides (8–10 amino acids
in leng h) ha a e eleased om he co po a ca diaca, a small neu oendoc ine gland
connec ed o he b ain [
11
,
12
]. They in e ac wi h a ious body sys ems in ol ed in
he ac i a ion o me abolism and ela ed p ocesses ( e iewed in [
11
–
13
]), which include
ac i a ion o diges i e enzymes [
14
–
16
], mobilisa ion o an i-oxida i e s ess esponse
( e iewed in [
13
]), and/o in e ac ions o AKHs wi h biogenic amines [
17
,
18
], among
o he s. I is also known ha some biogenic amines can con ol AKH p oduc ion [
17
].
None heless, i emains unclea whe he hese mechanisms a e in ol ed in AKH esponses
induced by na u al enoms.
In his s udy, he model Ame ican cock oach, Pe iplane a ame icana, was u ilised o mea-
su e he physiological e ec s o bee oxins. This is a e y popula model o physiological
and biochemical s udies and is p edominan ly used by Ame ican insec scien is s. The ho -
monal sys em o his species is well known and possesses wo AKHs called Pe am-CAH-I
(P. ame icana ca dioaccele a ing ho mone I (pGlu-Leu-Th -Phe-Th - P o-Asn-T p-NH
2
);
and Pe am-CAH-II (pGlu-Val-Asn-Phe-Se -P o-Asn-T p-NH
2
) [
19
]. Cock oaches a e no
he p ima y a ge s o bee a acks; howe e , he main oxic ac i i ies o bee enom by
molecules such as meli in o phospholipases a e no species-speci ic. Bee enom should
be e ec i e agains cock oaches since bees o en ace a acks by o he insec species. The
main goals o his s udy we e o desc ibe he e ec o bee oxins on se e al me abolic
cha ac e is ics and on muscle ul as uc u e. Fu he mo e, he possible ole o AKHs and
se e al o he biologically ac i e subs ances ( i ellogenins, dopamine, and glu a hione) in
de ence eac ions agains he enom was e alua ed.
2. Resul s
2.1. Gene al In e ac ions be ween Venom and AKH
Ini ial expe imen s in ol ed measu ing cock oach mo ali y 24 h a e applying in-
c easing doses o bee enom o de e mine he app op ia e dose (Figu e 1). A dose o 0.5 uL
enom pe cock oach was deemed he mos app op ia e since i elici ed app oxima ely 20%
mo ali y (LD50 = 1.063 uL) and p o ided a su icien physiological e ec whils lea ing a
sa is ac o y numbe o li ing insec s o he ollowing expe imen s. The es ima ed hal -li e
o 3.82 min o meli in was used o assess he a e a which bee enom is deg aded by he
cock oach de ence sys em in he haemolymph (Figu e 2).
Venom ea men elici ed se e e s ess in he cock oach body, since AKH le els in he
cen al ne ous sys em (CNS) signi ican ly ose by 3.8- old and 3.3- old a 6 h and 12 h,
espec i ely (Figu e 3). The le els e u ned o he con ol le el a e 24 h. Co esponding
AKH le els in he haemolymph signi ican ly inc eased by 3.6- old and 1.4- old a e 1 h
and 2 h, espec i ely (Figu e 4), indica ing ha AKH u no e in he haemolymph was
much as e han ha in he CNS. In e es ingly, he le el o AKHs in he CNS was in he
o de o pmols, while in haemolymph in he o de o mols.
Toxins 2022,14, 11 3 o 18
Toxins 2022, 13, x FOR PEER REVIEW 3 o 20
Figu e 1. The e ec o inc easing doses o honeybee enom on mo ali y o P. ame icana adul s 24 h
a e he ea men . Each poin ep esen s he mean ± SD; n = 3 g oups wi h 15–20 indi iduals in
each.
Venom ea men elici ed se e e s ess in he cock oach body, since AKH le els in
he cen al ne ous sys em (CNS) signi ican ly ose by 3.8- old and 3.3- old a 6 h and 12
h, espec i ely (Figu e 3). The le els e u ned o he con ol le el a e 24 h. Co esponding
AKH le els in he haemolymph signi ican ly inc eased by 3.6- old and 1.4- old a e 1 h
and 2 h, espec i ely (Figu e 4), indica ing ha AKH u no e in he haemolymph was
much as e han ha in he CNS. In e es ingly, he le el o AKHs in he CNS was in he
o de o pmols, while in haemolymph in he o de o mols.
2.2. E ec o Venom and AKH on Muscle Ul as uc u e
En enoma ion changed he muscle ul as uc u e in he cock oach ho ax (Figu e 5) by
inducing myo ib il des uc ion and gene al diso ganisa ion o o ganelles in muscle cells
(Figu e 5C). In e es ingly, co-applica ion wi h Pe am-CAH-II p e en ed his damage (Fig-
u e 5D). Meanwhile, ho mone adminis a ion alone (Figu e 5B) did no a ec he common
ul as uc u e o muscle cells compa ed wi h he Ringe saline con ol (Figu e 5A).
2.3. E ec o Venom and AKH on Selec ed Me aboli es
The le els o he main haemolymph nu ien s in he cock oach (ca bohyd a es, lipids,
and p o eins) we e modula ed by he enom ea men a e 6 h (Figu e 6), while he
changes we e less signi ican wi h inc easing ime (da a no shown). Ca bohyd a e le els
signi ican ly dec eased a e en enoma ion (1.8- old) and signi ican ly inc eased a e
Pe am-CAH-II ea men (1.8- old; Figu e 6A) as expec ed, since his ho mone p ima ily
mobilises ca bohyd a es in he cock oach body [20]. Co- ea men wi h bo h agen s elim-
ina ed hese luc ua ions and main ained ca bohyd a e le els a ha o he con ol. The
lipid le els signi ican ly inc eased in all expe imen al g oups a e he ea men s (Figu e
6B); howe e , hese changes we e sligh ly smalle han hose eco ded o ca bohyd a es
(1.3–1.7- old), wi h he o al lipid le el in he haemolymph app oxima ely wo imes lowe
han he o al ca bohyd a e le el. P o ein le els signi ican ly inc eased by 1.2–1.3- old ol-
lowing Pe am-CAH-II ea men alone o in combina ion wi h bee enom (Figu e 6C).
Figu e 1.
The e ec o inc easing doses o honeybee enom on mo ali y o P. ame icana adul s 24 h
a e he ea men . Each poin ep esen s he mean
±
SD; n= 3 g oups wi h 15–20 indi iduals in each.
Toxins 2022, 13, x FOR PEER REVIEW 4 o 20
Figu e 2. Tempo al changes in he amoun o meli in in he haemolymph o P. ame icana adul s
injec ed wi h 2 μL he honeybee enom. (A) Illus a i e HPLC eco d o meli in s anda d (1 μg).
(B) Illus a i e HPLC eco d o haemolymph ex ac (5 μL) 5 min a e enom ea men ; no peak
was obse ed in con ol haemolymph a RT = 10.3 min (da a no shown). (C) Le el o meli in in P.
ame icana adul haemolymph a di e en imes a e he ea men . The hal li e o 3.82 min was
es ima ed om plo s o log meli in i e. Each poin ep esen s he mean ± SD (n = 3–4).
Figu e 2.
Tempo al changes in he amoun o meli in in he haemolymph o P. ame icana adul s
injec ed wi h 2
µ
L he honeybee enom. (
A
) Illus a i e HPLC eco d o meli in s anda d (1
µ
g).
(
B
) Illus a i e HPLC eco d o haemolymph ex ac (5
µ
L) 5 min a e enom ea men ; no peak
was obse ed in con ol haemolymph a RT = 10.3 min (da a no shown). (
C
) Le el o meli in in
P. ame icana adul haemolymph a di e en imes a e he ea men . The hal li e o 3.82 min was
es ima ed om plo s o log meli in i e. Each poin ep esen s he mean ±SD (n= 3–4).
Toxins 2022,14, 11 4 o 18
Toxins 2022, 13, x FOR PEER REVIEW 5 o 20
Figu e 3. ELISA quan i ica ion o he Pe am-CAH-I and Pe am-CAH-II ( oge he —AKH) in CNS o
P. ame icana adul s a e he honeybee (0.5 μL) ea men ; esul s a e exp essed in pmol o Pe am-
CAH-II ( o explana ion see Sec ion 5.4). S a is ically signi ican di e ences be ween he enom
g oup and co esponding con ol (mean ± SD, n = 8) a each ime-poin e alua ed by S uden ’s - es
on 5% le el a e indica ed by * s a is ically signi ican di e ences among he ime-poin s a e he
enom ea men e alua ed by one-way ANOVA wi h Tukey’s pos - es a he 5% le el a e indi-
ca ed by di e en le e s abo e he columns (a,b.c) (no di e ences we e eco ded among he con-
ols). The numbe s abo e he columns ep esen old-di e ence o AKH le el as compa ed wi h
co esponding con ol.
Figu e 3.
ELISA quan i ica ion o he Pe am-CAH-I and Pe am-CAH-II ( oge he —AKH) in CNS o P.
ame icana adul s a e he honeybee (0.5
µ
L) ea men ; esul s a e exp essed in pmol o Pe am-CAH-II
( o explana ion see Sec ion 5.4). S a is ically signi ican di e ences be ween he enom g oup and
co esponding con ol (mean
±
SD, n= 8) a each ime-poin e alua ed by S uden ’s - es on 5% le el
a e indica ed by * s a is ically signi ican di e ences among he ime-poin s a e he enom ea men
e alua ed by one-way ANOVA wi h Tukey’s pos - es a he 5% le el a e indica ed by di e en le e s
abo e he columns (a,b.c) (no di e ences we e eco ded among he con ols). The numbe s abo e he
columns ep esen old-di e ence o AKH le el as compa ed wi h co esponding con ol.
2.2. E ec o Venom and AKH on Muscle Ul as uc u e
En enoma ion changed he muscle ul as uc u e in he cock oach ho ax (Figu e 5)
by inducing myo ib il des uc ion and gene al diso ganisa ion o o ganelles in muscle
cells (Figu e 5C). In e es ingly, co-applica ion wi h Pe am-CAH-II p e en ed his damage
(Figu e 5D). Meanwhile, ho mone adminis a ion alone (Figu e 5B) did no a ec he
common ul as uc u e o muscle cells compa ed wi h he Ringe saline con ol (Figu e 5A).
2.3. E ec o Venom and AKH on Selec ed Me aboli es
The le els o he main haemolymph nu ien s in he cock oach (ca bohyd a es, lipids,
and p o eins) we e modula ed by he enom ea men a e 6 h (Figu e 6), while he
changes we e less signi ican wi h inc easing ime (da a no shown). Ca bohyd a e le -
els signi ican ly dec eased a e en enoma ion (1.8- old) and signi ican ly inc eased a e
Pe am-CAH-II ea men (1.8- old; Figu e 6A) as expec ed, since his ho mone p ima -
ily mobilises ca bohyd a es in he cock oach body [
20
]. Co- ea men wi h bo h agen s
elimina ed hese luc ua ions and main ained ca bohyd a e le els a ha o he con ol.
The lipid le els signi ican ly inc eased in all expe imen al g oups a e he ea men s
(Figu e 6B); howe e , hese changes we e sligh ly smalle han hose eco ded o ca bo-
hyd a es
(1.3–1.7- old
), wi h he o al lipid le el in he haemolymph app oxima ely wo
imes lowe han he o al ca bohyd a e le el. P o ein le els signi ican ly inc eased by
1.2–1.3- old ollowing Pe am-CAH-II ea men alone o in combina ion wi h bee enom
(Figu e 6C).
Toxins 2022,14, 11 5 o 18
Toxins 2022, 13, x FOR PEER REVIEW 6 o 20
Figu e 4. HPLC quan i ica ion o he Pe am-CAH-I and Pe am-CAH-II ( oge he —AKHs) in hae-
molymph o P. ame icana adul s a e he honeybee (0.5 μL) ea men . (A) Illus a i e HPLC eco d
o AKH s anda ds Pe am-CAH-I and Pe am–CAH-II (10 and 10 pmol, espec i ely). (B) Illus a i e
HPLC eco d o an ex ac om 100 uL o haemolymph ( o de ails see Sec ion 5.4). (C) Le el o
AKHs in P. ame icana adul haemolymph a di e en imes a e he ea men . S a is ically signi i-
can di e ences be ween he enom g oup and co esponding con ol (mean ± SD, n = 3) a each
ime-poin e alua ed by S uden ’s - es on 5% le el a e indica ed by *; s a is ically signi ican di -
e ences among he ime-poin s a e he enom ea men e alua ed by one-way ANOVA wi h
Tukey’s pos - es a he 5% le el a e indica ed by di e en le e s abo e he columns (a,b,c) (no di -
e ences we e eco ded among he con ols). The numbe s abo e he columns ep esen old-di e -
ence o AKH le el as compa ed wi h co esponding con ol.
Figu e 4.
HPLC quan i ica ion o he Pe am-CAH-I and Pe am-CAH-II ( oge he —AKHs) in
haemolymph o P. ame icana adul s a e he honeybee (0.5
µ
L) ea men . (
A
) Illus a i e HPLC eco d
o AKH s anda ds Pe am-CAH-I and Pe am–CAH-II (10 and 10 pmol, espec i ely). (
B
) Illus a i e
HPLC eco d o an ex ac om 100 uL o haemolymph ( o de ails see Sec ion 5.4). (
C
) Le el o AKHs
in P. ame icana adul haemolymph a di e en imes a e he ea men . S a is ically signi ican di e -
ences be ween he enom g oup and co esponding con ol (mean
±
SD, n= 3) a each ime-poin
e alua ed by S uden ’s - es on 5% le el a e indica ed by *; s a is ically signi ican di e ences among
he ime-poin s a e he enom ea men e alua ed by one-way ANOVA wi h Tukey’s pos - es a
he 5% le el a e indica ed by di e en le e s abo e he columns (a,b,c) (no di e ences we e eco ded
among he con ols). The numbe s abo e he columns ep esen old-di e ence o AKH le el as
compa ed wi h co esponding con ol.
Toxins 2022,14, 11 6 o 18
Toxins 2022, 13, x FOR PEER REVIEW 7 o 20
Figu e 5. TEM pho os o he ho acic muscles om P. ame icana adul s 24 h a e ea men wi h (A)
Ringe saline (con ol), (B) 40 pmol Pe am-CAH-II, (C) 0.5 uL bee enom, and (D) 0.5 uL bee enom
+40 pmol Pe am-CAH-II oge he . Scale ba s in he Figs = 2 μm.
The ac i i y o he main diges i e enzymes in he cock oach midgu a e bee enom
applica ion showed some signi ican changes, bu hese we e gene ally small and ambig-
uous. The e o e, only he empo al e ec s o he enom, Pe am-CAH-II, and hei co-ad-
di ion a e shown (Figu e 7). Bee enom sligh ly s imula ed amylase ac i i y a e 6–24 h,
which was no modula ed by Pe am-CAH-II. The 2- old s imula ion using Pe am-CAH-II
alone was no obse ed un il 24 h a e ea men (Figu e 7A). Lipase ac i i y signi ican ly
inc eased by 2- old 24 h a e Pe am-CAH-II ea men , while o he ea men s had insig-
ni ican e ec s (Figu e 7B). P o ease ac i i y was signi ican ly s imula ed 12 h and 24 h
a e enom adminis a ion, while Pe am-CAH-II alone only s imula ed p o ease ac i i y
by 2.2- old a e 24 h. In e es ingly, Pe am-CAH-II s imula ion o all enzymes (24 h a e
ea men ) was signi ican ly elimina ed by he enom du ing co-applica ion o bo h agen s
(one-way ANOVA wi h Tukey’s pos - es : amylase, p = 0.0004; lipase, p = 0.0015; p o ease,
p = 0.002; da a no shown o cla i y).
Vi ellogenins a e ac i ely in ol ed in de ence eac ions agains a ious pa hogens
[9]. The e was a signi ican educ ion in bo h ~100 kDa i ellogenin p o eins by 8.2- old
and 2.9- old in he emale haemolymph 6 h a e enom adminis a ion (Figu e 8A,B).
Meanwhile, he esponse in he male haemolymph was less p onounced, wi h he hea ie
p o ein signi ican ly educed by 1.3- old and he ligh e p o ein una ec ed (Figu e 8A,C).
Figu e 5.
TEM pho os o he ho acic muscles om P. ame icana adul s 24 h a e ea men wi h
(
A
) Ringe saline (con ol), (
B
) 40 pmol Pe am-CAH-II, (
C
) 0.5 uL bee enom, and (
D
) 0.5 uL bee
enom +40 pmol Pe am-CAH-II oge he . Scale ba s in he Figs = 2 µm.
The ac i i y o he main diges i e enzymes in he cock oach midgu a e bee enom
applica ion showed some signi ican changes, bu hese we e gene ally small and am-
biguous. The e o e, only he empo al e ec s o he enom, Pe am-CAH-II, and hei
co-addi ion a e shown (Figu e 7). Bee enom sligh ly s imula ed amylase ac i i y a e
6–24 h,
which was no modula ed by Pe am-CAH-II. The 2- old s imula ion using Pe am-
CAH-II alone was no obse ed un il 24 h a e ea men (Figu e 7A). Lipase ac i i y
signi ican ly inc eased by 2- old 24 h a e Pe am-CAH-II ea men , while o he ea men s
had insigni ican e ec s (Figu e 7B). P o ease ac i i y was signi ican ly s imula ed 12 h
and 24 h a e enom adminis a ion, while Pe am-CAH-II alone only s imula ed p o ease
ac i i y by 2.2- old a e 24 h. In e es ingly, Pe am-CAH-II s imula ion o all enzymes (24 h
a e ea men ) was signi ican ly elimina ed by he enom du ing co-applica ion o bo h
agen s (one-way ANOVA wi h Tukey’s pos - es : amylase, p= 0.0004; lipase, p= 0.0015;
p o ease, p= 0.002; da a no shown o cla i y).
Vi ellogenins a e ac i ely in ol ed in de ence eac ions agains a ious pa hogens [
9
].
The e was a signi ican educ ion in bo h ~100 kDa i ellogenin p o eins by 8.2- old and
2.9- old
in he emale haemolymph 6 h a e enom adminis a ion (Figu e 8A,B). Mean-
while, he esponse in he male haemolymph was less p onounced, wi h he hea ie p o ein
signi ican ly educed by 1.3- old and he ligh e p o ein una ec ed (Figu e 8A,C).
Toxins 2022,14, 11 7 o 18
Toxins 2022, 13, x FOR PEER REVIEW 8 o 20
Figu e 6. The e ec o 0.5 μL honeybee enom and/o 40 pmol Pe am-CAH-II on (A) ca bohyd a e,
(B) lipid and (C) p o ein le els in he haemolymph o P. ame icana adul s 6 h a e he ea men .
S a is ically signi ican di e ences among he g oups (mean ± SD) e alua ed by one-way ANOVA
wi h Tukey’s pos - es a he 5% le el a e indica ed by di e en le e s abo e he columns (a,b,c) (n
= 6–8). The numbe s abo e he columns ep esen old-di e ence o he pa icula nu ien le el in
ea ed cock oaches compa ed wi h ha in con ols.
Bee enom applica ion insigni ican ly s imula ed dopamine le els in he cock oach
CNS 6 h a e ea men (Figu e 9). Ne e heless, a signi ican inc ease in his amine was
eco ded a e ea men wi h Pe am-CAH-II alone o oge he wi h enom by 2.3- old
and 2.4- old, espec i ely. Meanwhile, he le els o glu a hione oxida i e s ess ma ke s
( educed and oxidised glu a hione: GSH and GSSG, espec i ely) sligh ly inc eased ol-
lowing ea men wi h enom alone o ho mone alone (Figu e 10). Howe e , hei co-ad-
minis a ion led o a signi ican inc ease in GSH and GSSG le els by 5.3- old and 2.8- old,
espec i ely.
Figu e 6.
The e ec o 0.5
µ
L honeybee enom and/o 40 pmol Pe am-CAH-II on (
A
) ca bohyd a e,
(
B
) lipid and (
C
) p o ein le els in he haemolymph o P. ame icana adul s 6 h a e he ea men .
S a is ically signi ican di e ences among he g oups (mean
±
SD) e alua ed by one-way ANOVA
wi h Tukey’s pos - es a he 5% le el a e indica ed by di e en le e s abo e he columns (a,b,c)
(n= 6–8)
. The numbe s abo e he columns ep esen old-di e ence o he pa icula nu ien le el in
ea ed cock oaches compa ed wi h ha in con ols.
Toxins 2022,14, 11 8 o 18
Toxins 2022, 13, x FOR PEER REVIEW 9 o 20
Figu e 7. The empo al e ec o 0.5 uL honeybee enom and/o 40 pmol Pe am-CAH-II on (A) am-
ylase, (B) lipase and (C) p o ease ac i i ies in he midgu o P. ame icana adul s 6, 12 and 24 h a e
he ea men . S a is ically signi ican di e ences be ween he con ol and enom g oups, be ween
he con ol and Pe am-CAH-II g oups, and be ween he con ol and bo h agen s g oups (all mean ±
SD) e alua ed by one-way ANOVA wi h Dunne ’s pos - es a he 5% le el a e indica ed by *, @
and #, espec i ely (n = 6–8). The numbe s abo e he columns ep esen old-di e ence o he pa -
icula enzyme ac i i y in ea ed cock oaches compa ed wi h ha in con ols. Fo u he s a is ics
see he co esponding ex .
Figu e 7.
The empo al e ec o 0.5 uL honeybee enom and/o 40 pmol Pe am-CAH-II on (
A
)
amylase, (
B
) lipase and (
C
) p o ease ac i i ies in he midgu o P. ame icana adul s 6, 12 and 24 h a e
he ea men . S a is ically signi ican di e ences be ween he con ol and enom g oups, be ween he
con ol and Pe am-CAH-II g oups, and be ween he con ol and bo h agen s g oups (all mean
±
SD)
e alua ed by one-way ANOVA wi h Dunne ’s pos - es a he 5% le el a e indica ed by *, @ and #,
espec i ely (n= 6–8). The numbe s abo e he columns ep esen old-di e ence o he pa icula
enzyme ac i i y in ea ed cock oaches compa ed wi h ha in con ols. Fo u he s a is ics see he
co esponding ex .
Bee enom applica ion insigni ican ly s imula ed dopamine le els in he cock oach
CNS 6 h a e ea men (Figu e 9). Ne e heless, a signi ican inc ease in his amine was
eco ded a e ea men wi h Pe am-CAH-II alone o oge he wi h enom by 2.3- old
and 2.4- old, espec i ely. Meanwhile, he le els o glu a hione oxida i e s ess ma k-
e s ( educed and oxidised glu a hione: GSH and GSSG, espec i ely) sligh ly inc eased
ollowing ea men wi h enom alone o ho mone alone (Figu e 10). Howe e , hei
co-adminis a ion led o a signi ican inc ease in GSH and GSSG le els by 5.3- old and
2.8- old, espec i ely.
Toxins 2022,14, 11 9 o 18
Toxins 2022, 13, x FOR PEER REVIEW 10 o 20
Figu e 8. The e ec o 0.5 uL honeybee enom on i ellogenin le el in P. ame icana adul haemo-
lymph 6 h a e he ea men . (A)—illus a i e 4–20% SDS polyac ylamide gel ( emales 0.4 uL and
males 1 uL haemolymph/well: 1— emale con ol, 2— emale en enomed, 3—male con ol, 4—male
en enomed; 5—molecula s anda ds); (B)— i ellogenin quan i ica ion in emales; (C)— i ellogenin
quan i ica ion in males. S a is ically signi ican di e ences be ween he enom g oups and co e-
sponding con ols e alua ed by S uden ’s - es on 5% le el a e indica ed by * (n = 6–8). The numbe s
abo e he columns ep esen old-di e ence o he pa icula i ellogenin le el in ea ed cock-
oaches compa ed wi h ha in con ols.
Figu e 9. The e ec o 0.5 uL honeybee enom and/o 40 pmol Pe am-CAH-II on dopamine le els
in he b ain o P. ame icana adul s 6 h a e he ea men . S a is ically signi ican di e ences among
he g oups (mean ± SD) e alua ed by one-way ANOVA wi h Tukey’s pos - es a he 5% le el a e
Figu e 8.
The e ec o 0.5 uL honeybee enom on i ellogenin le el in P. ame icana adul haemolymph
6 h a e he ea men . (
A
)—illus a i e 4–20% SDS polyac ylamide gel ( emales 0.4 uL and males 1 uL
haemolymph/well: 1— emale con ol, 2— emale en enomed, 3—male con ol, 4—male en enomed;
5—molecula s anda ds); (
B
)— i ellogenin quan i ica ion in emales; (
C
)— i ellogenin quan i ica ion
in males. S a is ically signi ican di e ences be ween he enom g oups and co esponding con ols
e alua ed by S uden ’s - es on 5% le el a e indica ed by * (n= 6–8). The numbe s abo e he columns
ep esen old-di e ence o he pa icula i ellogenin le el in ea ed cock oaches compa ed wi h
ha in con ols.
Toxins 2022, 13, x FOR PEER REVIEW 10 o 20
Figu e 8. The e ec o 0.5 uL honeybee enom on i ellogenin le el in P. ame icana adul haemo-
lymph 6 h a e he ea men . (A)—illus a i e 4–20% SDS polyac ylamide gel ( emales 0.4 uL and
males 1 uL haemolymph/well: 1— emale con ol, 2— emale en enomed, 3—male con ol, 4—male
en enomed; 5—molecula s anda ds); (B)— i ellogenin quan i ica ion in emales; (C)— i ellogenin
quan i ica ion in males. S a is ically signi ican di e ences be ween he enom g oups and co e-
sponding con ols e alua ed by S uden ’s - es on 5% le el a e indica ed by * (n = 6–8). The numbe s
abo e he columns ep esen old-di e ence o he pa icula i ellogenin le el in ea ed cock-
oaches compa ed wi h ha in con ols.
Figu e 9. The e ec o 0.5 uL honeybee enom and/o 40 pmol Pe am-CAH-II on dopamine le els
in he b ain o P. ame icana adul s 6 h a e he ea men . S a is ically signi ican di e ences among
he g oups (mean ± SD) e alua ed by one-way ANOVA wi h Tukey’s pos - es a he 5% le el a e
Figu e 9.
The e ec o 0.5 uL honeybee enom and/o 40 pmol Pe am-CAH-II on dopamine le els
in he b ain o P. ame icana adul s 6 h a e he ea men . S a is ically signi ican di e ences among
he g oups (mean
±
SD) e alua ed by one-way ANOVA wi h Tukey’s pos - es a he 5% le el a e
indica ed by di e en le e s abo e he columns (a,b) (n= 6–8). The numbe s abo e he columns ep esen
old-di e ence o he pa icula dopamine le el in ea ed cock oaches compa ed wi h ha in con ols.
Toxins 2022,14, 11 16 o 18
ans e ime: 100.0
µ
s, and p e-pulse s o age: 10.0
µ
s. Fo MS/MS a mul iple eac ion
moni o ing mode was used wi h he ollowing pa ame e s: o all p ecu so ions: m/z
154.091, 308.089, and 613.160 Da, he isCID ene gy: 20.0 eV, and collision ene gy: 35 eV.
5.10. Da a P esen a ion and S a is ical Analyses
Da a was analysed using P ism e sion 6.0 (G aphPad So wa e, San Diego, CA,
USA). Ba g aphs ep esen mean
±
s anda d de ia ion (SD) and he numbe s o epli-
ca es (n) is depic ed in he igu e legends. S a is ical di e ences we e e alua ed by S u-
den ’s - es (Figu es 3,4and 8), and by one-way ANOVA wi h he Tukey’s pos - es
(Figu es 3,4,6,7,9and 10) and wi h Dunne ’s pos - es (Figu e 7).
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h ps://www.mdpi.com/a icle/
10.3390/ oxins14010011/s1, Table S1: Lis o plan s blooming in he icini y o he apia y in ˇ
Ceské
Budˇejo ice (CZ; 48◦58031.92400 N, 14◦26044.67100 E; al i ude 390 m) in June 2020.
Au ho Con ibu ions:
Concep ualiza ion, D.K.; Me hodology, K.B., J. ˇ
C., H.Š. and R.G.; Valida ion,
D.K. and O.Z.; Fo mal Analysis, D.K. and O.Z.; In es iga ion, D.K.; Resou ces, D.K., R.G. and O.Z.;
Da a Cu a ion, D.K. and O.Z.; W i ing—D.K.; W i ing—Re iew & Edi ing, all au ho s. All au ho s
ha e ead and ag eed o he published e sion o he manusc ip .
Funding:
This s udy was suppo ed by p ojec RVO 60077344 o he Ins i u e o En omology, and he
p ojec CEITEC 2020 (LQ1601).
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : No applicable.
Acknowledgmen s:
This s udy was suppo ed by he p ojec s RVO 60077344 o he Ins i u e o
En omology (K.B., J. ˇ
C., H.Š. and D.K.). R.G. and O.Z. we e suppo ed by he p ojec CEITEC 2020
(LQ1601). The pho os o insec s in he G aphical Abs ac we e aken by M. Hejníko á.
Con lic s o In e es :
The au ho s decla e no con lic o in e es . The sponso s had no ole in he
design, execu ion, in e p e a ion, o w i ing o he s udy.
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