Detailed proteome mapping of newly emerged honeybee worker hemolymph and comparison with ...

14 Pages·2017·3.86 MB·English

Detailed proteome mapping of newly emerged honeybee
worker hemolymph and comparison with the red-eye
pupal stage
Tomas Erban, Karel Harant, Martin Kamler, Martin Markovic, Dalibor Titera
To cite this version:
Tomas Erban, Karel Harant, Martin Kamler, Martin Markovic, Dalibor Titera. Detailed proteome
mappingofnewlyemergedhoneybeeworkerhemolymphandcomparisonwiththered-eyepupalstage.
Apidologie, 2016, 47 (6), pp.805-817. ￿10.1007/s13592-016-0437-7￿. ￿hal-01532447￿
HAL Id: hal-01532447
https://hal.archives-ouvertes.fr/hal-01532447
Submitted on 2 Jun 2017
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Apidologie(2016)47:805–817 Original article
*INRA,DIBandSpringer-VerlagFrance,2016
DOI:10.1007/s13592-016-0437-7
Detailed proteome mapping of newly emerged honeybee
worker hemolymph and comparison with the red-eye
pupal stage
TomasERBAN1,KarelHARANT2,MartinKAMLER3,MartinMARKOVIC1,DaliborTITERA3
1LaboratoryofProteomics,CropResearchInstitute,Drnovska507/73,Praha6-RuzyneCZ-16106,Czechia
2FacultyofScience,CharlesUniversityinPrague,Prague,Czechia
3BeeResearchInstituteatDol,LibcicenadVltavou,Czechia
Received18September2015–Revised24January2016–Accepted15February2016
Abstract– The honeybee,Apismellifera, undergoescompletemetamorphosisbefore transitioning tothe adult
stage.Thenewlyemergedindividualandthered-eyepupastagearewelldefinedandeasilyrecognizableinthetime
lifecyclehoneybeeand,therefore,veryusefulforstudyingphysiologicalanddevelopmentalfactors.Weanalyzedin
detailthehemolymphproteomeofnewlyemergedhoneybeeworkerusing2D-E-MS/MS(pI3-10and4-7).The
comparisonofidenticalhemolymphvolumes(20μLper2D-E)fornewlyemergedbeeandred-eyepuparevealeda
dramaticdecreaseinthenumberofspots(qualitativechanges)andoverallproteinquantityduringthenon-feeding
stage.Theresultsincreaseourknowledgeabouthoneybeemetamorphosisduringthenon-feedingperiodandclarify
previous findings regarding particular proteins. The results will be useful for future comparative physiological,
developmental,andhost-pathogenstudiesonindividualorpopulationlevel.
Apismellifera /hemolymph/metamorphosis/hexamerin/ferritin
1. INTRODUCTION honeybee life cycle and, therefore, very useful
for studying physiological and developmental fac-
Thehoneybee,Apismellifera L.,isaholome- torsthatcanbecausedbyaging,entomopathogens
tabolousinsectthatundergoescompletemetamor- (such as viruses), bacteria, Nosema, the
phosisbeforetransitioningtotheadultstage.The hemolymph-suckingmiteVarroa,ortheinfluence
developmenttimeofEuropeanhoneybeeworkers ofpesticides.
istypically21days.Thepupalstagebeginswith Similarly to human blood, insect hemolymph
themoltfromthelastlarvalstage,afewdaysafter containsimportantindicatorsofthephysiological
a brood cell was sealed. During this period, the stateofthebody.Sincetheproteincompositionof
body isreconstructed and, finally, the capping is the honeybee hemolymph varies between devel-
eatenawayandtheadultemerges(Winston1987). opmentalstages,hemolymphproteinscanbeused
The just/newly emerged honeybee, like the red- toinvestigatecastedifferencesanddevelopmental
eyepupastage(Erbanetal.2014),isawell-defined, changes (Chan et al. 2006; Chan and Foster
easily recognizable developmental stage in the 2008). Changes in the hemolymph protein com-
positionfromlarvaltopupaldevelopmentstages
ElectronicsupplementarymaterialTheonlineversionof ofA.melliferaligustica werestudiedbyWoltedji
thisarticle(doi:10.1007/s13592-016-0437-7)contains etal.(2013),andaproteomicapproachhasbeen
supplementarymaterial,whichisavailabletoauthorized usedtostudyhemolymphproteomechangescom-
users.
paringA.mellifera andA.cerana duringworker
Correspondingauthor:T.Erban,[email protected] brooddevelopment,includingpupae (Fengetal.
ManuscriptEditor:KlausHartfelder 2014). We previously described the hemolymph
806 T.Erbanetal.
of the red-eye pupal stage at the proteome level were obtained from Carl Roth (Karlsruhe, Germany).
(Erbanetal.2014).Inaddition,honeybeehemo- DeStreak Rehydration solution(CatNo. 17-6003-19),
lymphproteinsofdifferentdevelopmentalstages IPGbufferpH3–10(CatNo.17-6000-87),IPGbuffer
were detected in the ectoparasite Varroa pH 4–7 (Cat No. 17-6000-86), ammonium persulfate
destructor (Erbanetal.2015). (APS;CatNo.17-1311-01),Immobiline™DryStrippH
In this study, we analyzed in detailthe hemo- 3–10, 13 cm (Cat No. 17-6001-14), Immobiline Dry
lymphofnewlyemergedhoneybeeworkersusing Strip pH 4–7, 13 cm (Cat No. 17-6001-13), and
two-dimensionalgelelectrophoresis(2D-E)com- PhastGel™BlueR(CatNo.17-0518-01)wereobtained
bined with a matrix-assisted laser desorption/ from GE Healthcare Life Sciences (GE) (Uppsala,
ionization (MALDI) time-of-flight (TOF)/TOF Sweden).Trypsin(CatNo.v5111)wasobtainedfrom
proteomicsapproach.Inadditiontotheproteomic Promega(Fitchburg,WI,USA).
2D-E-MS/MS mapping of emerged honeybees,
weaimedtocomparetheseresultswiththehemo-
lymph protein composition of the red-eye pupal 2.2. Biologicalsamples
stagedescribedinourpreviousstudy(Erbanetal.
2014). The results indicated that at the end of The newly emerged honeybees used in this
metamorphosis/timeofemergence,thehoneybee study were collected from monitored Varroa-free
hemolymphbecamedepletedofproteinsandonly A. mellifera carnica colony at the Bee Research
afewproteinsremainedatahigherconcentration. Institute at Dol in Czechia. For hemolymph col-
A dramatic change in the hemolymph protein lection, individual honeybees were removed with
compositionwasidentifiedwhencomparingred- forceps as they were chewing through the cell
eyepupaewith newlyemerged bees. Inaddition caps. Thus, the bees did not eat before collection,
to these comparative results, we believe that the nor were they affected by factors associated with
descriptionofthemethodicalapproachpresented the environment outside the hive. On the other
here,i.e.,simpledirectproteomicanalysiswithout hand, the bees were not influenced by long ab-
sample processing producing quality 2D-Es or sence of the queen or hive because we collected
comparison of same hemolymph volumes be- the samples within 20 min after the comb was
tween stages, will also be of interest for further taken from the hive. Hemolymph was immediately
studies. retrieved from the dorsal vessels using a glass
capillary, after puncturing the abdominal interseg-
2. MATERIALSANDMETHODS mental membrane between the 4th and 5th ter-
gites. Approximately 20–40 μL of transparent he-
2.1. Reagents molymph was collected from each individual. The
hemolymph was transferred from the glass capil-
Iodoacetamide(IAA;CatNo.57670),dithiothreitol laries to microcentrifuge tubes and immediately
(DTT;CatNo.43817),agarose(CatNo.A7431),am- frozen on dry ice, or mixed in volumes of either
monium bicarbonate (ABC; Cat No. A6141), 20 or 30 μL with DeStreak Solution containing
trifluoroaceticacid(TFA;CatNo.91707),acetonitrile 0.5 % IPG buffer pH 3–10 or 4–7 and then frozen
(ACN; Cat No. 34998), 37.5:1 acrylamide/bis- on dry ice. The samples were stored at −80 °C in
acrylamide solution (Cat No. 01709), N,N,N′,N′- a deep freezer (Thermo, Waltham, MA, USA)
tetramethylethylenediamine (TEMED; Cat No. until use. Prior to preparation of this paper, we
T9281), Bradford reagent (Cat No. B6916), methanol performed more than 50 2D-E separations
LC-MSCHROMASOLV®(CatNo.34966),α-cyano- (Coomassie or fluorescence stained and 13- or 7-
4-hydroxycinnamicacid(CHCA;CatNo.70990),and cm strips; data not shown) of emerged worker bee
HPLC/MS-grade water were obtained from Sigma- hemolymph from different hives. The 2D-E im-
Aldrich(St.Louis,MO,USA).Aceticacidwasobtain- ages presented in this study were collected in
edfromLach-ner(Neratovice,Czechia).Ultra-pureso- April and originated from a single colony. Six
dium dodecyl sulfate (SDS; Cat No. 2326.2), glycine 2D-Es were selected for protein identifications in
(CatNo.3908.2),andTrisbase(TRIS;CatNo.4855.3) this study.
Hemolymphproteomeofnewlyemergedhoneybees 807
2.3. Two-dimensionalgelelectrophoresis proteins had highest scores for some other insects.
Hits scored with P <0.05 were considered
Atotalof20or30μLofhemolymphfromasingle significant.
honeybeewasusedforeach2D-Eexperiment.Wedid
notprocessthehemolymphbeforeanalysis,analogous
2.5. Semiquantitative evaluation of 2D-Es
to Erban et al. (2014), as the samples mixed with
ofemergedbeeswithred-eyepupa
DeStreakcontaining0.5%IPGbuffer(pH3–10or4–
7)weredirectlyusedforisoelectrofocusing(IEF).IEF
Tocomparethe2D-Ehemolymphofemergedhon-
wasperformedonanEttanIPGphor3(GE).Thesepa-
eybeeswiththepupalstage,weadoptedtheresultsfrom
ration was performed in 13-cm ceramic strip holders
ourpreviousstudy(Erbanetal.2014)ofred-eyepupal
andImmobilinedrystrips(pH3–10or4–7).Thesep-
hemolymphperformedonthesameapparatususingthe
aration program was as follows: (1) step, 30 V, 10 h
same methods and identical 20-μL hemolymph vol-
(activerehydration);(2)step,500V,500Vh;(3)grad,
umes.Becausethepatternsofthe2D-Esoftheemerged
1000V,800Vh;(4)grad,6000V,15,000Vh;and(5)
beesandred-eyepupaewerefoundtobeverydifferent,
step,6000 V, 16,000Vh.The IEFranfor 19 hand a
weevaluatedthedataatasemiquantitativelevelonly.
total of 32,600 Vh. Following IEF, the strips were
Thedataarepresentedasthepresenceorabsenceofa
equilibratedfor15mininanequilibrationbuffercon-
proteinintherespectivedevelopmentalstage,orlower,
tainingDTT,followedby15mininabufferwithIAA.
higher, or of similar abundance. Dymension software
Thestripswereplacedovera14%SDS-PAGEgeland
(ver. 3.0.1.4; Syngene, Cambridge, UK) was used to
fixed with 1 % agarose. Electrophoresis was run at a
determinethenumberofspotsandanormalizedvolume
constant voltageinacooledapparatus(SE600Ruby,
oftheIdgf4proteinspotinthe2D-Esinthetwodevel-
GE). The gels were fixed (40 % methanol, 10 % ice
opmentalstages.Thesettingsofthespotanalyzerwere
acetic acid, and 50 % ddH O) overnight and stained
2 as follows: (i) Spot Analysis: Gaussian limit thresh-
using0.02%PhastGelBlueR.Unusedfixingsolution
old—0.05; minimum Gaussian quality index—0.1,
was used for destaining. The results were visualized
minimumsignificance—100,maximumGaussiansper
withtheG:BOX(Syngene,Cambridge,UK).
patch—3, minimum fit probability—0.01; (ii) Patch
Analyzer: blur radius—1.2, detection confidence ra-
2.4. MALDI TOF/TOF protein
tio—30, separation confidence ratio—10, peak limit
identification
threshold—0.01, splitting threshold—0.02; and (iii)
Gaussiandetector:maximumdistancesquaredtocen-
Spots (0.5- to 1-mm inner diameter) were se-
ter—1.5,minimumradius.
lected from the Coomassie-stained gels and sub-
jected to 4800 Plus MALDI TOF/TOF (Applied
Biosystems/MDS SCIEX; Foster City, CA, USA) 3. RESULTS
analysis. The sample preparation procedures and
subsequent protein identification of trypsin digests 3.1. Results of 2D-E mapping of newly
followed the methodology described by Erban emergedworkerbeehemolymph
et al. (2014). The spectra were searched against
the non-redundant (nr) NCBI database using Representative images of the analyzed 20
MASCOT 2.2 (Boston, MA, USA). The database or 30 μL of hemolymph volumes of 2D-E
search criteria were as follows: enzyme, trypsin; Coomassie-stained, 14 % SDS-PAGE, pI 3–
taxonomy, Insecta (749,851 sequences); fixed 10, and 4–7 IEF gels are presented in
modification, carbamidomethylation (C); variable Figures 1 and 2a–c, respectively; see also
modifications, deamidated (NQ), methionine oxi- the Supplemental Material Tables S1 and S2
dation (M); protein mass, unrestricted; peptide for the list of identified proteins, with details
mass tolerance, ±60–100 ppm; fragment mass tol- on identification and characterization, respec-
erance, ±0.3 Da; and one missed cleavage tively. The use of a pI 4–7 IEF separation in
allowed. For presentation of data, the taxonomy 2D-E analyses greatly improved the separa-
Insecta was selected due to the fact that some tion of accumulated protein spots, enabling
808 T.Erbanetal.
Figure1.Representative2D-EimageofapI3–10,14%SDS-PAGEanalysiswithmarkedidentifiedproteinspots
of honeybee worker hemolymph collected at the time of emergence. The 2D-E image results from a 30-μL
hemolymphanalysisofasingleindividual.SeeTableS1andS2forthelistofidentifiedproteinswithdetailsof
MS/MSidentifications.SeealsoFigureS1forthe2D-EfigurespI3–10on14%SDS-PAGEgeneratedfroma20-
μLhemolymph;FigureS1showsthreebiologicalreplicates.
us to easily distinguish isoforms of the Blast searches and the position of ferritin spots
proteins. in2D-E(Figure2c).
Significant (P <0.05) MALDI TOF/TOF re-
sults were protein scores greater than 71. 3.2. Results of the comparison of 2D-Es of
Identifications of some proteins had highest newly emerged worker bees and red-
scores to species different from A. mellifera, eyepupae
e.g.,glutathione S-transferase sigma class or fer-
ritin.Proteinidentificationstoferritinheavychain The analysis via the Dymension software
[A. cerana cerana] (JF330112.1; gi|357372779) demonstrated a low amount in the number of
and ferritin light chain [A. cerana cerana] spots and decreased protein abundance in the
(JF340051.1; gi|357372804) apparently corre- emerged bee hemolymph compared to the
spond to A. mellifera ferritin light and heavy red-eye pupal hemolymph (Table I). The
chains, respectively. This was confirmed by number of visible hemolymph protein spots
Hemolymphproteomeofnewlyemergedhoneybees 809
Figure2.Representative2D-EimageofapI4–7,14%SDS-PAGEanalysiswithmarkedidentifiedproteinspotsof
honeybeeworkerhemolymphcollectedatthetimeofemergence.a The2D-Eimagewasgeneratedfrom30μLof
hemolymph of a single individual.b Details on high molecular weight proteins from 20 μL of hemolymph. c
Detailsshowingisoformsofferritinheavychain(FerHCH)andferritinlightchain(FerLCH).SeeTableS1andS2
forthelistofidentifiedproteinswithdetailsabouttheMS/MSidentifications.
was 4.54 times lower in the newly emerged and S2 for detail) was used to infer on
bee compared to the red-eye pupa, as indi- quantitative changes via a normalized volume
cated by the consensus spots detected in the (Table I). The normalized volume of the
2D-E (Table I). Chitinase-like protein Idgf4 Idgf4 spot was 4.48 times higher in emerged
and its isoforms had the same quantity in bee hemolymph compared to red-eye pupa.
both stages. Therefore, the spot (spot no. Comparing the 2D-E pI 3–10, 14 % SDS-
L021 in Figure 1) corresponding to Idgf4 in PAGE of 20-μL hemolymph of newly emerged
the samples of the two stages (see Figure S1 bees with 20-μL red-eye pupal hemolymph
810 T.Erbanetal.
TableI.Consensusspotsandnormalizationvolumesofselectedchitinase-likeproteinIdgf4spotsdeterminedby
Dymensionsoftware.
Sampleno./stage Consensusspots Norm.vol.ofIdgf4spot
1/emergedbee 387 1.677
2/emergedbee 317 1.696
3/emergedbee 367 1.643
Average±SDof1–3 357±36 1.672±0.027
4/red-eyepupa 1383 0.436
5/red-eyepupa 1669 0.296
6/red-eyepupa 1807 0.388
Average±SDof4–6 1620±216 0.373±0.071
Threeindependentbiologicalreplicatesfrombothred-eyepupaeandnewlyemergedbeeswerealigned;seeFiguresS1andS2for
thefiguresusedintheanalysisandademonstrationoftheanalysisusingDymension.Chitinase-likeproteinIdgf4wasfoundtobe
constantinbothstages.Theresultsshoweda4.54-folddecreaseinthenumberofspotsaftertransitionfromthered-eyepupalstage
tothenewlyemergedbee.The4.48-foldlowernormalizedvolumeoftheIdgf4spotinred-eyepupalhemolymphcomparedto
emergedbeehemolymphindicatedacorrespondingdecreaseinproteininthehemolymph
(Figure 3, TableII ), clearly different 2D-E pat- independently, and at the same respective posi-
terns wereobtained.The proteinspotswereana- tion, the results matched. Some major proteins
lyzedusingMALDI TOF/TOFinthe twostages werenotdetectedinthe2D-Eofnewlyemerged
Figure3.Comparisonof2D-Eproteomemaps(pI3–10;14%SDS-PAGE)generatedfrom20μLhemolymphof
newlyemergedworkerbees(a)andred-eyepupae(b).The2D-Eimageofthered-eyepupalstagewasreproduced
fromourpreviousstudy(Erbanetal.2014).Selectedproteinsaremarkedinthefigure.SeeTableIIforthelistof
proteingroupsmarked.SeeFigureS1forthe2D-Efiguresofthethreebiologicalreplicatesgeneratedfrom20μLof
newlyemergedbeehemolymphandFigureS2forthe2D-Efiguresofthethreebiologicalreplicatesgeneratedfrom
20μLofred-eyepupalhemolymph.
Hemolymphproteomeofnewlyemergedhoneybees 811
TableII.Semiquantitativeevaluationofmajorandselectedchangesbetweenred-eyepupalandnewlyemergedbee
hemolymph.
No. Proteinname Newly Red-eye Semiquantitative
emergedbee pupa neb/rep
1 Ferritin Yes Yes Higher
2 GSTsigmaclass Yes Yes Lower
3 Chitinase-likeproteinIdgf4-like Yes Yes Similar
4 Hexamerin70a Yes Yes Similar
5 Toll-like Yes No –
6 Alphaglucosidase Yes Yes Similar
7 LOC413792;fibrillin;peroxisomalN(1)-acetyl-spermine/ Yes No –
spermidineoxidase-likeisoform
8 Actin Yes Yes Lower
9 ApolipophorinIII Yes Yes Similar
10 NPC2homolog Yes Yes Similar
11 OBP14 Yes Yes Similar
12 Chemosensoryprotein3precursor Yes Yes Higher
13 OBP13 Yes Yes Similar
14 Apolipophorins Yes Yes Similar
15 Hexamerin70c No Yes –
16 Hexamerin70b No Yes –
17 Hexamerin110 No Yes –
18 Beta-ureidopropionase No Yes –
19 Heatshockproteincognate4 Yes Yes Lower
20 Heatshockproteincognate3 No Yes –
21 Heatshockproteincognate5 No Yes –
22 Heatshockproteinbeta-1 Yes Yes Lower
23 Short-chaindehydrogenase/reductase No Yes –
24 Peroxiredoxin-likeprotein No Yes –
25 FABP-likeprotein Yes Yes Lower
26 Phospholipidhydroperoxideglutathioneperoxidase Yes Yes Similar
27 Circadianclock-controlledprotein Yes No –
SeeFigure3forthecomparisonofmarkedproteingroupsinthe2D-Es.Legend:Yes—theproteinwasdetectedin2D-Esofthe
stage;No—theproteinwasdetectedin2D-Esofthestage;dash(–)—semiquantitativeevaluationwasnotapplicableastheprotein
wasnotdetectedinonetypeoftheexaminedsamples=qualitativechange
bees compared to red-eye pupae, or vice versa hemolymph is low in protein, probably due to
(TableII). extensiveproteindepletion(metabolicuse)during
the non-feeding period associated with body re-
4. DISCUSSION construction.Therefore,theproteinsatthetimeof
emergence represent a minimal need before the
Comparison of both the consensus spots and firstfeedingallowsthesynthesisofnewproteins.
normalized volume of the selected Idgf4 spot Somemajorproteinswerenotdetectedinthe2D-
demonstrated considerable protein depletion at Eofnewlyemergedworkerscomparedtored-eye
the time of a bee’s emergence compared to the pupae, e.g., a short-chain dehydrogenase reduc-
red-eye pupal stage. At emergence, the tase;storagehexamerins110,70b,and70c;beta-
812 T.Erbanetal.
ureidopropionase; and some heat shock proteins 4.2. Hexamerin—storage or transport
orperoxiredoxin-likeproteins.Ontheotherhand, protein?
someproteinsweredetectedsolelyinthe2D-Eof
emerged bees, e.g., a circadian clock-controlled Hexamerins are principally storage proteins
proteinandtoll-likereceptor13.Thus,thechang- thatserve as a source of energyandaminoacids
esinhemolymphproteinsarelargelyataqualita- in non-feeding periods. Overall, four hexamerin
tive level. The presence of proteins at similar genes, Hex70a, Hex70b, Hex70c, and Hex110,
abundance (hexamerin 70a, Idgf4) indicated its wereidentifiedinthehoneybeegenome(Martins
equalneedinthered-eyepupaeandinthenewly etal.2010).Proteinproductsofthesegeneswere
emergentworkerbee.Theproteincompositionof identifiedinthehemolymphofdifferentstagesof
thehemolymphofnewlyemergedbees,i.e.,rela- thehoneybeevia1D-E(Dantyetal.1998;Cunha
tively high levels of ferritin, glutathione S-trans- etal.2005;MartinsandBitondi2012).Allofthe
ferase,ortoll-likereceptor13,indicatesprecondi- fourhexamerinshavebeenidentifiedby2D-Ein
tioning to new stimuli, i.e., stress from a new red-eyepupalhemolymph(Erbanetal.2014).In
environmentorpathogen,afterleavingthecapped the 2D-E analyses of newly emerged honeybee
comb,whichisaclosedenvironment.Themajor workers, wesuccessfullyidentifiedproteinprod-
groups of proteins detected in our analysis are uctsofonlyhexamerin70a.Thedisappearanceof
discussed below; the remainder of the detected hexamerins110,70b,and70cindicatesthatthey
proteinsislistedinTableS1. exertedafunctionduringmetamorphosis,whileat
thetimeofemergence,theirfunctionended.
According to Martins et al. (2010), all of the
4.1. Chitinase-likeproteinIdgf4isconstant fourgenesarehighlytranscribedbythelarvalfat
body, while only 70a and 110 have detectable
Idgf4 is considered a developmental pro- expression in the adult fat body. According to
tein in A. mellifera larva (Li et al. 2010; Cunha et al. (2005), a four-band hexamerin pat-
Feng et al. 2014). Feng et al. (2014) found ternfoundinthelarvalstageismaintainedwith-
Idgf4 to be up-regulated in the hemolymph outapparentmodificationuntilthe middleofthe
of both A. mellifera and A. cerana from the pupalstage,whenmelaninpigmentsappearinthe
larval to the pupal stage (Feng et al. 2014). thoracic cuticle, thus characterizing the light
Relatively strong spots of Idgf4 were identi- pigmented pupal phase; the hexamerin 110 band
fied in winter (Erban et al. 2013) and sum- becomes barely visible, whereas the hexamerin
mer (Bogaerts et al. 2009) honeybee hemo- 70a, 70b, and 70c subunits were still perfectly
lymph. Even though Idgf4 is apparently pres- detectable. Subsequently, i.e., at the transition to
ent (used) throughout the life cycle of hon- adultlife, hexamerins 70b and70c progressively
eybee workers, its exact functions remain decrease in the hemolymph, but they were still
unknown. In Drosophila, Idgfs stimulate the visibleatthe7thdayofadultlife.Theabundance
proliferation, polarization, and motility of ofthehexamerin70asubunitdoesnotapparently
imaginal disc cells in cooperation with insu- change, at least until the 7th day of adult life
lin (Kawamura et al. 1999). According to (Cunha et al. 2005). Danty et al. (1998) showed
Butler et al. (2003), Idgf4 is expressed in thathexamerin70aistheonlyhexamerinpresent
the peripodial membrane (primarily in dorsal in a large amount in hemolymph of later adult
cells) and, presumably, the secretion of Idgf4 honeybee worker stages. Hexamerins 70b and
influences the development of the columnar 70c exhibit a similar developmental profile be-
epithelium (Butler et al. 2003). The constant cause they disappear from the hemolymph with
concentration of Idgf4 indicated its equal worker emergence, while hexamerin 110 disap-
need in the red-eye pupa and in the emerged pearsearlierthan70band70c(Dantyetal.1998).
worker hemolymph. Moreover, Idgf4 could Bitondi et al. (2006) found that hexamerin 110
be used as a quantitative reference for other subunits are highly abundant in larval hemo-
protein spots. lymph, but are decreased at the larval spinning
Hemolymphproteomeofnewlyemergedhoneybees 813
stage,andremainatlowlevelsinpupaeandadults 4.4. GlutathioneS-transferasesigmaclass
(Bitondietal.2006).
Weconcludethatourfindingsusing2D-Epro- In general, glutathione S-transferases (GSTs)
teomic analysis clarify previous results that the are enzymes connected to the antioxidative de-
hexamerins 110, 70b, and 70c are depleted from fensesystemthateliminatesreactiveoxygenspe-
the honeybee worker hemolymph at the time of ciesgeneratedasby-productsofaerobicmetabo-
emergence, while only hexamerin 70a remains lism (Corona and Robinson 2006). Sigma GSTs
present (functional) at a higher concentration. haveevolvedspecializedfunctionsandhaveboth
Thecontinuedpresenceofhexamerin70ainhon- anti- and pro-inflammatory functions, depending
eybeehemolymphatthetimeofemergenceindi- on the type of immune response (Flanagan and
cates that it is not used primarily as a storage Smythe2011).SigmaGSTshavebeendetectedin
proteinbutexertsanotherfunctionandmayserve different honeybee developmental stages, show-
atransportfunction. ingthatthisenzymeisubiquitousthroughoutthe
entire life cycle of the honeybee (Erban et al.
4.3. Ferritin—a high abundance protein in 2014). The pivotal role as a protectant against
newlyemergedhoneybees theby-productsofoxidativestressduringhoney-
bee metamorphosis has been suggested (Erban
rOur results indicate that isoforms assigned et al. 2014). The abundance of Sigma GST par-
as NCBInr record A. mellifera PREDICTED: tially decreased compared with pupal hemo-
ferritin heavy chain (XP_624076.1; lymph. However, the still considerable presence
gi|66524161) are of higher molecular weight suggests an apparent importance of the enzyme
than those that were identified as NCBInr fortheemerginghoneybee.Apparently,theseen-
record A. mellifera PREDICTED: ferritin zymes play an important, whether protective or
subunit (XP_624044.3; gi|328782011). Thus, not,roleintheemerginghoneybee,aswellasin
the honeybee ferritins are present in the H thepupa.
and the L type. We showed that the ferritin
level at the time of emergence increased in 4.5. Olfaction—Odorant-binding and
hemolymph compared with pupal hemo- chemosensoryproteins
lymph. In vertebrates, elevated serum ferritin
levels have been reported in a number of Chemosensory proteins (CSPs) are similar to
pathological states (Morikawa et al. 1995), odorant-bindingproteins(OBPs)andsharesome
and ferritin acts as an immunosuppressor, of their characteristics, but CSPs belong to an
signaling molecule and immune regulator evolutionarilydistinctlineage(Foretetal.2007).
(Recalcati et al. 2008). In Drosophila, over- We detected Obp13, Obp14, and CSP3 in the
expression of both the HCH and LCH genes hemolymphofnewlyemergedworkerbees,sim-
is required to confer protection against oxi- ilartothatfromred-eyepupae(Erbanetal.2014).
dative stress (Missirlis et al. 2007). At the WhiletheabundanceofOBPswassimilarinboth
end of metamorphosis and at the time when stages,CSP3increasedatthetimeofemergence.
the honeybee adult leaves the stable environ- Furthermore, Fenget al. (2014) detected Obp13,
ment of the comb cell, the individual is put Obp14,andCSP3inhemolymphofA.mellifera
under increased stress, and the immune sys- andA.cerana bees.Zhengetal.(2011)detected
tem will start to adapt to the new environ- Obp13 and 14 during A. mellifera pupal head
ment. Therefore, it is possible that the high development. Obp13, 14, 15, and chemosensory
level of ferritin in the emerged honeybee protein 3 (ASP3) were identified in the hemo-
hemolymph is an important component of lymph of A. mellifera summer workers
the immune system and may regulate the (Bogaerts et al. 2009), and Obp14 was found in
honeybee immune system so as not to over- winter worker hemolymph (Erban et al. 2013).
react to the new stimulus outside the brood Briandetal.(2002)observedthatCSP3isabrood
cell. pheromone carrier in A. mellifera. Foret et al.