research article ovarian development and...

Research ArticleOvarian Development and Vitellogenin Gene Expression underHeat Stress in Silkworm Bombyx mori

Satinath Paul and Bela Keshan

Department of Zoology North-Eastern Hill University Shillong Meghalaya 793022 India

Correspondence should be addressed to Bela Keshan bkeshanhotmailcom

Received 1 April 2016 Revised 19 July 2016 Accepted 27 July 2016

Academic Editor Vladimir Kostal

Copyright copy 2016 S Paul and B Keshan This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Thepresent study observed the effect of heat stress on ovarian development fecundity and vitellogenin gene expression in silkwormBombyx mori The result showed that the heat shock treatment to spinning larvae and pupae at 39∘C (1 h and 2 h) did not cause anyadverse effect on the reproductive performance of B moriHowever the heat shock treatment at 42∘C or above caused a decrease inthe fecundityTheheat shock treatment to day 2 pupae for 2 h at 45∘Ccaused a drastic effect on the development of ovary asmeasuredby gonadosomatic index The study thus showed that a brief exposure of Bombyx larvae and pupae to a temperature of 42∘C orhigher much prevalent in tropical countries like India greatly affects the ovarian development and reproductive performanceof this commercially important insect The study further showed a developmental- and tissue-specific expression of vitellogeninmRNA in fat body and ovary upon heat shock When heat shock treatment was done at 39∘C and 42∘C to spinning larvae ovaryshowed an upregulation in the expression of vitellogenin mRNA whereas fat body failed to do so However at 45∘C both fat bodyand ovary showed a downregulation The heat shock treatment to day 2 pupae showed an upregulation in the vitellogenin mRNAexpression in both fat body and ovary even at 45∘C The upregulation in the expression of vitellogenin upon heat shock indicatesits role in thermal protection of Bombyx larvae and pupae

1 Introduction

Global warming is one of themajor challenges for the survivaland reproduction of many life forms including insects Theincrease of global mean surface temperature is likely to be03ndash48∘C by the end of 21st century relative to 1986ndash2005[1] It is warned that the heat waves will occur with a higherfrequency and a longer duration [1] Studies have shownthat warmer temperatures associated with climate change canpotentially affect insect speciesrsquo population dynamics directlythrough effects on survival generation time fecundity anddispersal [2 3] Insects being ectothermic organisms arevery likely to respond quickly to increased temperatures [4]However the response of individual insect specieswill dependon their geographical range trophic level and natural history[3] For majority of temperate insect species increasedtemperature will allow them to extend their geographicalrange and enhance their population fitness [2] However fortropical insects increased temperature is likely to have the

most deleterious consequences as tropical insects are rela-tively sensitive to temperature change and are currently livingvery close to their optimal temperature [5] Temperatureabove the optimum temperature is perceived as heat stress byall living organisms

In a tropical country like India environmental factorssuch as temperature and humidity play a major role in thesuccess of sericulture industry Because of the extensive andcareful domestication over centuries the silkworm Bombyxmori is verymuch susceptible to abrupt temperature changesStudies have shown that cells of all known organisms includ-ingBmori respond to stress such as temperature by increasedsynthesis of heat shock or stress proteins (Hsps) [6ndash10]Accumulation of these stress proteins induced by exposureto mild stress results in a transient state of stress resistanceIt is indicated that heat inducible Hsps are important forheat induced hormesis in longevity and heat stress resistancein Drosophila [11] Hormesis is the phenomenon where amild exposure to an otherwise detrimental factor such as

Hindawi Publishing CorporationPsycheVolume 2016 Article ID 4242317 8 pageshttpdxdoiorg10115520164242317

2 Psyche

heat insecticides and radiation becomes beneficial for manyorganism and life history traits [12ndash15] In Drosophila asignificant increase in lifespan of females repeatedly exposedto mild heat stress through hormesis has been suggestedby Hercus et al [16] In the oriental fruit moth Grapholitamolesta the exposure of females to a single heat event at 38∘Cfor 4 h caused an increase in the lifespan and length of theoviposition period leading to a potential increase in lifetimefecundity and suggesting hormesis [17] However Forbes [18]suggested that not all aspects of organism performance canbe hormetic simultaneously and there might be an energetictrade-off among life history traits Since both survival andreproduction require energy and since energy is limited itis expected that there will be a trade-off between these twofitness components [19]

In B mori although literatures are available on the effectof heat stress on survival and expression of heat shockproteins [8 20ndash25] little information is available on the effectof heat stress on its reproductive fitness It is suggested thatthough the increased expression of heat shock proteins undermild heat stress plays an important role in helping insect tosurvive its high level usually brings negative physiologicaleffects on organisms [26] The improved thermotoleranceat the cost of reduced reproductive performance has beenobserved inmany insect species such asBicyclus anynana andHelicoverpa armigera [27ndash30] In Spodoptera litura and Neo-seiulus barkerimild thermal stress increases thermotolerancebut brings a detrimental effect on fertility and fecundity[26 31]

The developing ovary in Bombyx consists of four poly-trophic meroistic ovarioles each of which contains a longlinear array of developing follicles in progressively advancingstage of development Each follicle is surrounded by a layerof follicle cells and contains an oocyte and interconnectednurse cells Vitellogenin a precursor of major egg yolkprotein plays an important role in promoting the growthand differentiation of developing oocytes The regulation ofvitellogenin is directly under the control of hormones atthe transcriptional level [32] In Bombyx its biosynthesisis regulated transcriptionally in a sex- and stage-dependentmanner [33] Vitellogenin functions not only as an energyreserve for the developing embryo but also in innate immuneresponse [34] Singh et al [35] showed the antibacterialactivity of vitellogenin and found that the infected silkwormlarvae treated with purified vitellogenin survived the full lifecycle in contrast to untreated animals Increased vitellogeninexpression in Caenorhabditis elegans potently increases theirresistance against pathogenic bacteria and heat [36] RecentlyIhle et al [37] showed that aging and lifespan in honeybees are affected by vitellogenin This protein influencesworker lifespan both as a regulator of behavioral matura-tion and through antioxidant and immune functions as anexperimental reduction of vitellogenin expression resulted indecreased lifespan and increased susceptibility to oxidativedamage

Since development time and reproductive output of aninsect are nearly as important to population growth as thesurvival of individuals the present study has been carriedout to investigate the consequences of heat stress on ovarian

development and fecundity as well as on vitellogenin geneexpression in B mori

2 Material and Methods

21 Insect Silkworm (Bombyx mori) L (CSR 18 a bivoltinerace) was used for present study Disease-free layings ofsilkworms breed CSR 18 were obtained from Central Ser-icultural Germplasm Resource Centre CSB Hosur TamilNadu and reared with fresh mulberry leaves in laboratoryunder controlled conditions (temperature 25plusmn1∘C humidity60ndash90 photoperiod 12L 12D) Larvae and pupae werestaged according to days after ecdysis and the availablemorphological markers such as spinneret pigmentation andgut purge

22 Heat Shock Treatment For heat shock treatment day 3spinning larvae and day 2 pupae were placed individually inglass test tubes plugged with cotton and were submerged in awater bath at 39∘C 42∘C and 45∘C for 1 h and 2 h respectivelyas described [38] After the treatment the larvae and pupaewere returned to controlled laboratory conditions Femalelarvae and pupae were considered for heat shock treatmentMale silkworm larvae and pupaewere subjected to heat shocktreatment only for the purpose of copulation

23 Determination of Gonadosomatic Index Heat shock wasgiven to day 2 female pupae and developing ovaries weredissected out after 48 h (day 4) 96 h (day 6) and 144 h(day 8) of heat shock treatment The isolated ovaries wererinsed with insect ringer solution blotted dry with filterpaper and the weight was measured using digital weighingbalanceGonadosomatic index (GSI)was calculated using theequation GSI = ovary wet weight (g)total body weight (g) times100 [39] The experiments were repeated 2-3 times and meanvalue of GSI was calculated for each experimental group fromthe data obtained from 6ndash8 individuals

24 Estimation of Fecundity Day 3 spinning larvae and day 2pupae (both males and females) were subjected to heat shocktreatment and the adult moths were collected for matingThe moths were allowed to copulate for 5 h and afterwardsthe moths were decoupled and the females were kept foroviposition and males were disposed off The fecundity wascalculated by counting the number of eggs laid by each femalewithin 24 h time period The experiments were repeated 3times and the mean value of fecundity was calculated fromthe data obtained from 10ndash12 individuals

25 RNA Isolation and Semiquantitative RT-PCR The fatbody and ovary were dissected out from 5th instar larvae andpupae under cold insect ringer solution and homogenizedimmediately in TRIzol reagent (Invitrogen) and stored atminus70∘C Total RNA was isolated according to the manufac-turerrsquos protocol The concentration of RNA was measuredspectrophotometrically at 260 nm The purity of the totalRNA was assessed using A

260280ratios Denaturing agarose

Psyche 3

C 39-1 39-2 42-1 42-2 45-1 45-200

05

10

15

20

25

30

35

NSNSNS

Day 4

NS

C 39-1 39-2 42-1 42-2 45-1 45-20

3

6

9

12

15

18

21

24NS

Day 6

NS

C 39-1 39-2 42-1 42-2 45-1 45-2

NS

Day 8

NS

Gon

ados

omat

ic in

dex

(times100

)

lowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

Gon

ados

omat

ic in

dex

(times100

)

Gon

ados

omat

ic in

dex

(times100

)

0369

1215182124273033363942

Figure 1 Effect of heat stress on gonadosomatic index (ovary) Heat shockwas given to pupae 2 days after larval-pupal ecdysis and developingovarioles were dissected out frompupae on days 4 6 and 8 from each experimental groupThewet weight of developing ovaries wasmeasuredafter rinsing with insect ringer solution and gonadosomatic index was calculated with respect to total body weight Each histogram barrepresents the mean gonadosomatic index (119899 = 6ndash8) and the error bar represents standard error of the mean (SEM) The asterisks show thedata which are significantly different from control sample (unpaired 119905-test lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001) NS not significant at119901 lt 005 C control 39-1 heat shock at 39∘C for 1 h 39-2 heat shock at 39∘C for 2 h 42-1 heat shock at 42∘C for 1 h 42-2 heat shock at 42∘Cfor 2 h 45-1 heat shock at 45∘C for 1 h 45-2 heat shock at 45∘C for 2 h

gel electrophoresis was performed to ascertain the integrityof isolated RNA

The cDNA was synthesized from total RNA using M-MLV reverse transcriptase (Invitrogen) and oligo dT 18primers (Integrated DNATechnologies Inc) followingman-ufacturerrsquos protocol The cDNA was subsequently used foramplification of target DNA by Polymerase Chain Reaction(PCR) using the gene specific forward and reverse primersForward and reverse primers for vitellogenin and riboso-mal protein (rp49) were designed based on the sequencedata available on Genbank For vitellogenin (Accessionnumber NM 001043844) the forward primer and reverseprimers are 51015840-GCTTTGCCTAGGACCCTACG-31015840 and 51015840-GCAGCGGACTTAAAAGCAACC-31015840 and for rp49 (Acces-sion number AB048205) the forward primer and reverseprimers are 51015840-GCATCAATCGGATCGCTATGAC-31015840 and

51015840-CAAGAAGACCCGTCATATGCT-31015840 Briefly the sampleswere heated at 95∘C for 10min followed by 30 amplificationcycles (95∘C for 30 s 60∘C for 30 s and 72∘C for 45 s) anda final 10 minutes extension period at 72∘C The amplifiedproducts were subjected to 35 agarose gel electrophoresisand molecular size was confirmed by running DNA stan-dard Images of the RT-PCR ethidium bromide-stained gelwere acquired and quantification of the band densities wasperformed using Image-J program The experiments wererepeated 2-3 times with 2-3 independent biological replicatesin each experiment For data normalization rp49was used asa reference gene The transcript level of the vitellogenin genewas calculated relative to rp49 expression for each sampleby comparing the band densities The normalized value ofvitellogenin mRNA expression level in one of the controlsamples was designated as 1 and afterwards the relative

4 Psyche

C 39-1 39-2 42-1 42-20

100

200

300

400

500

600

NS NS NSNS

Num

ber o

f egg

s lai

dfe

mal

e

Larval spinning stagePupal stage

NS

lowastlowastlowast

Figure 2 Effect of heat stress on fecundity Heat shock was givento spinning larvae and day 2 pupae and number of eggs laid perfemale was counted from each experimental group Each histogrambar represents the mean value for fecundity (119899 = 10ndash12) andthe error bar represents standard error of the mean (SEM) Theasterisks show the data which are significantly different from controlsample (unpaired 119905-test lowast119901 lt 001) The abbreviations used are asmentioned for Figure 1

expression levels for the remaining control samples and theexperimental samples were calculated Mean and SEM wereobtained using relative expression level values for control andall experimental groups

26 Statistical Analysis Studentrsquos 119905-test was performed for theanalysis of the data using GraphPad Prism 6 software

3 Results

31 Effect of Heat Stress on Ovarian Development and GSIGonadosomatic index is an important indicator to showthe gonadal developmental progress and its maturity In thepresent study Bombyx ovary showed an increase in GSI withthe advancement of ovarian development during pupal stageThe GSI increased from 28 plusmn 02 to 216 plusmn 04 from day 4to day 6 of pupal life (Figure 1) On day 8 GSI increased to356 plusmn 13 The mild heat shock treatment to day 2 pupae at39∘C (1 h and 2 h) failed to show any adverse effect on GSIHowever the heat shock treatment at 42∘C and 45∘C causeda severe effect on GSI The heat shock exposure at 42∘C (1 hand 2 h) caused a significant decrease (119901 lt 005ndash001) in GSIas observed on day 6 and day 8 of pupal life (Figure 1) Theheat shock treatment at 45∘C showed a more severe effectthan 42∘C and even day 4 pupae showed a decrease in GSI(Figure 1) With 1 h exposure at 45∘C a decrease in GSI to126 plusmn 07 and 153 plusmn 08 was observed on day 6 and day8 respectively (Figure 1) With the increase of heat shockduration to 2 h at 45∘C a drastic decrease in GSI to 32 plusmn 023and 40 plusmn 02 was observed on day 6 and day 8 of pupal liferespectively (Figure 1)

32 Effect of Heat Stress on Fecundity The heat shock treat-ment to either spinning larvae or day 2 pupae at 39∘C failedto bring any adverse effect on the fecundity of the adult femalemoth (Figure 2) However the heat shock treatment at 42∘Ccaused a significant decrease in fecundity The heat shockexposure to day 2 pupae for 1 h at 42∘C caused a decrease infecundity to 421 plusmn 20 from 491 plusmn 12 observed for untreatedadult moth (Figure 2) A 2 h exposure at this temperatureshowed a further decrease in fecundity to 387 plusmn 24 Forspinning larvae the heat shock treatment at 42∘C for 1 hdid not show any significant changes in the fecundity of thesurvived adult However the exposure to spinning larvaefor 2 h at this temperature caused a significant decrease infecundity to 418plusmn14 (Figure 2)The fecundity data could notbe measured for experimental group heat stressed at 45∘C asthis treatment caused death of the most of the heat stressedindividual although the heat stressed pupae could survive forfew days as evident by a response to a stimulus like touch[25]

33 Effect of Heat Stress on Vitellogenin mRNA ExpressionThe heat shock treatment to spinning larvae and pupaeshowed a developmental- and tissue-specific expression ofvitellogenin mRNA in fat body and ovary The heat shocktreatment to spinning larvae at mild and mild-to-severe heatstressed condition (39∘C and 42∘C) showed a significantincrease in vitellogenin mRNA expression level but onlyin ovary (Figure 3(b)) and not in fat body (Figure 3(a))However at severe-to-lethal heat stressed condition (45∘C)ovary showed a significant decrease in vitellogenin expression(Figure 3(b)) The fat body tissues also showed a decrease invitellogenin expression at this temperature but only for a 2 hexposure (Figure 3(a))

The heat shock treatment to day 2 pupae showed an unex-pected but a very promising result on vitellogenin mRNAexpression level In both fat body and ovary an increase invitellogeninmRNAexpressionwas observed uponheat shocktreatment even at severe-to-lethal heat stressed condition(45∘C) (Figures 4(a) and 4(b)) It should be noted that theheat stressed pupae at 45∘C could survive for many days afterthe heat shock treatment although they could not emergeas adults In day 4 pupae however vitellogenin expressionin both fat body and ovary tissues decreased to almostcontrol level except at 45∘C 2 h where a downregulation invitellogenin expression was observed (Figure 4(c))

4 Discussion

Our previous study [25] showed an increased expression ofheat shock protein 90 (hsp90) in Bombyx larvae and pupaeupon mild heat shock treatment at 39∘C The study furthersuggested that the upregulation of hsp90 at a particular heatshock temperature is associated with the ability of the animalto survive upon heat stress The present study showed thatheat shock to Bombyx larvae and pupae at mild temperature(39∘C) is without any adverse effect on ovarian developmentand fecundityThus the ability of silkworm larvae and pupaeto withstand heat stress at mild temperature apparently with

Psyche 5

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12NS

NSNS NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowast

(a)C 39-1 39-2 42-1 42-2 45-1 45-2

00

04

08

12

16

20

24

28

32

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

-vg

-rp49

45-245-142-242-139-239-1C

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

(b)

Figure 3 Vitellogenin mRNA expressions in spinning larvae upon heat shock (a) Fat body (b) OvaryThe upper panel showed expression ofvitellogenin and rp49 as revealed by semiquantitative RT-PCRThe lower panel showed relative expression level of vitellogeninThe transcriptlevel of the vitellogenin gene was calculated relative to rp49 expression for each sample Each histogram bar represents the mean relativeexpression of vitellogenin (119899 = 5ndash7) and the error bar represents standard error of the mean (SEM) The asterisks show the data whichare significantly different from control sample (unpaired 119905-test lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001) The abbreviations used are asmentioned for Figure 1

no reproductive cost seems to be positively related with anincreased expression of hsp90 Sarup et al [40] suggestedthat the mild heat shock treatment might induce hormeticresponse as this treatment upregulates the synthesis of Hspsand extends the lifespan in Drosophila

The heat shock treatment to Bombyx larvae and pupae ata temperature of 42∘ was not lethal for their survival andmajority of them after heat shock treatment entered intonormal developmental program [25] The heat shock at thistemperature also induced the expression of hsp90 [25] Thepresent study showed that the heat shock exposure to Bom-byx larvae and pupae at this temperature affects their ovariandevelopment and reproductive performance as evident fromdecreased gonadosomatic index and fecundity A ldquotrade-offrdquothus exists between thermal resistance and reproduction inB mori In D melanogaster repeated mild heat stress causedincreased thermotolerance but also brought a detrimentaleffect on fertility and fecundity [16] In D virilis heat stressresults in oocyte maturation delays degradation of earlyvitellogenic egg chambers and inhibition of yolk proteingene expression in follicle cells and accumulation of matureoocytes [41] Heat shock also had deleterious effects on ovar-ian development in Tribolium castaneum [42] on fecundityinTrialeurodes vaporariorum and Liriomyza huidobrensis [2743] and on differentiation of the apyrene spermatozoa inBombyx [44]

The vitellogenin mRNA showed a tissue- anddevelopmental-specific expression upon heat shock treat-

ment during larval spinning and pupal stages In larvalovary the heat shock at 39∘C and 42∘C caused an increasein vitellogenin mRNA expression but with the increase ofheat shock temperature to 45∘C a downregulation in itsexpression was observed This pattern of upregulation ofvitellogenin mRNA expression in larval ovary followed thesame trend as was observed for hsp90mRNAexpression [25]The induction of vitellogenin mRNA expression in larvalovary upon mild heat stress when the larvae could surviveand molted to pupae indicates that vitellogenin might havea protective role in the survival of B mori and thus suggestsits role in hormesis like hsp90 The increased expressionof vitellogenin might also help the differentiating tissueslike ovary at spinning stage to cope up with the challengedsituation raised because of the heat shock treatment

The data on the increased expression of vitellogeninmRNA upon heat shock treatment even at 45∘C during pupalstage further support its protective action It has to be notedthat when heat shock was given at a temperature of 45∘Cthe pupae could survive for many days although it could noteclose An upregulation in the expression of vitellogenin withan increased magnitude of heat shock treatment is difficult toexplain now but a detailed mechanistic study in future canexplain the differential expression of vitellogenin upon heatshock It is also difficult to hypothesize that the inductionof vitellogenin upon heat shock treatment involves insecthormones although it is quite known that the vitellogenesisin insects including B mori is under the direct regulation of

6 Psyche

00

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

45-245-142-242-139-239-1C(a)

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(b)

C 39-1 39-2 42-1 42-2 45-1 45-200

02

04

06

08

10

12

14

NSNSNSNS NSNSNSNS

NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

Fat bodyOvary

NS

lowastlowastlowast

(c)

Figure 4 VitellogeninmRNAexpressions in pupae (a) Fat body day 2 pupae (b)Ovary day 2 pupae (c) Fat body and ovary day 4 pupaeTheupper panel showed expression of vitellogenin and rp49 as revealed by semiquantitative RT-PCRThe lower panel showed relative expressionlevel of vitellogenin as normalized with rp49 transcript level Each histogram bar represents the mean relative expression of vitellogenin(119899 = 5ndash7) and the error bar represents standard error of the mean (SEM) The asterisks show the data which are significantly different fromcontrol sample (unpaired 119905-test lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001) The abbreviations used are as mentioned for Figure 1

insect hormones like juvenile hormone andor ecdysteroidsThus the possibility remains that heat shock might have dis-rupted the normal hormonal milieu in Bombyx and therebychanged the vitellogenin expression in the fat body and ovaryTeixeira and polavarapu [45] showed that heat stress inhibitsthe completion of pupal diapause in Rhagoletis mendax andsuggested that the failure to complete the development wasdue to the damage of the endocrine system by heat stress as

the application of exogenous 20-hydroxyecdysone on thesepupae caused them to develop to the adult stage

The data further suggests that vitellogenin expression istemperature-dependent as in pupae after the first signif-icant increase as observed within 1 h of heat shock treat-ment its expression returned to normal expression levelat 48 h of heat shock treatment except at 45∘C where adownregulation in its expression was observed Thus the

Psyche 7

temperature-dependent variations in the expression of vitel-logenin indicate its role in thermal protection of silkwormlarvae and pupae besides its classical role as a precursor ofegg yolk proteins Although information regarding the otherfunctional aspects of vitellogenin is not available much butlimited literature showed its hemagglutinating and antibac-terial activities [34] The antibacterial activity of vitellogeninhas been demonstrated in Bombyx and it is shown thatinfected silkworm larvae treated with vitellogenin survivedthe full life cycle in contrast to untreated animals [35]Vitellogenin also has the protective role against oxidativestress It is synthesized at high levels in honey bee queens andis abundant in long-lived worker protecting the sterile honeybee workers from oxidative stress [46] Increased vitellogeninexpression in C elegans potently increases their resistanceagainst heat [36] Ihle et al [37] showed that an experimentalreduction of vitellogenin expression in honey bees resultedin decreased lifespan and increased susceptibility to oxidativedamage

In conclusion the study hypothesizes that vitellogeninmight have a protective role on the survival of silkwormFurther a brief exposure of Bombyx larvae and pupae to atemperature of 42∘C or higher (which is very common intropical countries) greatly affects the ovarian developmentand reproductive performance of this commercially impor-tant insect Further studies can be undertaken to know themechanism of regulation of vitellogenin expression underheat stress in B mori and other organisms and its protectiverole by interfering with its synthesis

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This study was supported by Department of Science andTechnology (DST) Government of India a project underSERC FAST Track Scheme (SRFTLS-0262008) for YoungScientist sanctioned to Bela Keshan Thanks are to theDirector Central Sericultural Germplasm Resource Centre(CSGRC) CSB Hosur Tamil Nadu India for providing tothe authors disease-free layings (dfls) of silkworm

References

[1] Intergovernmental Panel on Climate Change (IPCC) ClimateChange Synthesis Report Summary for Policymakers 2014httpswwwipccchpdfassessment-reportar5syrAR5 SYRFINAL SPMpdf

[2] J S Bale G J Masters I D Hodkinson et al ldquoHerbivoryin global climate change research direct effects of risingtemperature on insect herbivoresrdquo Global Change Biology vol8 no 1 pp 1ndash16 2002

[3] E E Stange and M P Ayres ldquoClimate change impacts insectsrdquoin eLS John Wiley amp Sons Chichester UK 2010

[4] C Robinet and A Roques ldquoDirect impacts of recent climatewarming on insect populationsrdquo Integrative Zoology vol 5 no2 pp 132ndash142 2010

[5] C A Deutsch J J Tewksbury R B Huey et al ldquoImpactsof climate warming on terrestrial ectotherms across latituderdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 105 no 18 pp 6668ndash6672 2008

[6] M E Feder and G E Hofmann ldquoHeat-shock proteins molec-ular chaperones and the stress response evolutionary andecological physiologyrdquo Annual Review of Physiology vol 61 pp243ndash282 1999

[7] L-H Huang H-S Wang and L Kang ldquoDifferent evolutionarylineages of large and small heat shock proteins in eukaryotesrdquoCell Research vol 18 no 10 pp 1074ndash1076 2008

[8] J Li S H H Moghaddam X Du B-X Zhong and Y-Y ChenldquoComparative analysis on the expression of inducible HSPs inthe silkworm Bombyx morirdquoMolecular Biology Reports vol 39no 4 pp 3915ndash3923 2012

[9] J G Soslashrensen T N Kristensen and V Loeschcke ldquoTheevolutionary and ecological role of heat shock proteinsrdquo EcologyLetters vol 6 no 11 pp 1025ndash1037 2003

[10] L Zhao and W A Jones ldquoExpression of heat shock proteingenes in insect stress responsesrdquo Invertebrate Survival Journalvol 9 pp 93ndash101 2012

[11] J G Soslashrensen T N Kristensen K V Kristensen and VLoeschcke ldquoSex specific effects of heat induced hormesis inHsf-deficient Drosophila melanogasterrdquo Experimental Gerontologyvol 42 no 12 pp 1123ndash1129 2007

[12] J R Cypser P Tedesco and T E Johnson ldquoHormesis and agingin Caenorhabditis elegansrdquo Experimental Gerontology vol 41no 10 pp 935ndash939 2006

[13] E Le Bourg and N Minois ldquoIncreased longevity and resistanceto heat shock in Drosophila melanogaster flies exposed tohypergravityrdquo Comptes Rendus de l Academie des Sciences Serie3 Sciences de la Vie vol 320 pp 215ndash221 1997

[14] N Minois ldquoLongevity and aging beneficial effects of exposureto mild stressrdquo Biogerontology vol 1 no 1 pp 15ndash29 2000

[15] P A Parsons ldquoHormesis an adaptive expectation with empha-sis on ionizing radiationrdquo Journal of Applied Toxicology vol 20no 2 pp 103ndash112 2000

[16] M J Hercus V Loeschcke and S I S Rattan ldquoLifespanextension of Drosophila melanogaster through hormesis byrepeated mild heat stressrdquo Biogerontology vol 4 no 3 pp 149ndash156 2003

[17] L N Liang W Zhang G Ma A A Hoffmann C Ma andR N Guedes ldquoA single hot event stimulates adult performancebut reduces egg survival in the oriental fruit moth Grapholithamolestardquo PLoS ONE vol 9 no 12 Article ID e116339 2014

[18] V E Forbes ldquoIs hormesis an evolutionary expectationrdquo Func-tional Ecology vol 14 no 1 pp 12ndash24 2000

[19] P Calow and R M Sibly ldquoA physiological basis of populationprocesses ecotoxicological implicationsrdquo Functional Ecologyvol 4 no 3 pp 283ndash288 1990

[20] O Joy and K P Gopinathan ldquoHeat shock response in mulberrysilkworm races with different thermotolerancesrdquo Journal ofBiosciences vol 20 no 4 pp 499ndash513 1995

[21] V B Chavadi A H Sosalegowda and M H BoregowdaldquoImpact of heat shock on heat shock proteins expression bio-logical and commercial traits of Bombyx morirdquo Insect Sciencevol 13 no 4 pp 243ndash250 2006

[22] H B Manjunatha R K Rajesh and H S Aparna ldquoSilkwormthermal biology a review of heat shock response heat shockproteins and heat acclimation in the domesticated silkwormBombyxmorirdquo Journal of Insect Science vol 10 article 204 2010

8 Psyche

[23] A H Sosalegowda R R Kundapur and M H BoregowdaldquoMolecular characterization of heat shock proteins 90 (HSP83)and 70 in tropical strains of Bombyx morirdquo Proteomics vol 10no 15 pp 2734ndash2745 2010

[24] V K Rahmathulla C M K Kumar B S Angadi and VSivaprasad ldquoAssociation of climatic factors on populationdynamics of leaf roller Diaphania pulverulentalis hampson(Lepidoptera Pyralidae) in mulberry plantations of sericultureseed farmrdquo Psyche vol 2012 Article ID 186214 6 pages 2012

[25] B Keshan S Paul T Bembem and S D Kh ldquoTissue-specificexpression patterns of heat shock protein 90 transcripts insilkworm Bombyx Morirdquo Journal of Entomology and ZoologyStudies vol 2 no 6 pp 53ndash59 2014

[26] Y Shen Y-J Gong J Gu L-H Huang and Q-L FengldquoPhysiological effect of mild thermal stress and its induction ofgene expression in the common cutworm Spodoptera liturardquoJournal of Insect Physiology vol 61 no 1 pp 34ndash41 2014

[27] L-H Huang B Chen and L Kang ldquoImpact of mild tem-perature hardening on thermotolerance fecundity and Hspgene expression in Liriomyza huidobrensisrdquo Journal of InsectPhysiology vol 53 no 12 pp 1199ndash1205 2007

[28] T L Geister MW Lorenz M Meyering-Vos K H Hoffmannand K Fischer ldquoEffects of temperature on reproductive outputegg provisioning juvenile hormone and vitellogenin titres inthe butterfly Bicyclus anynanardquo Journal of Insect Physiology vol54 no 8 pp 1253ndash1260 2008

[29] G K Mironidis and M Savopoulou-Soultani ldquoEffects of heatshock on survival and reproduction of Helicoverpa armigera(Lepidoptera Noctuidae) adultsrdquo Journal of Thermal Biologyvol 35 no 2 pp 59ndash69 2010

[30] S A Janowitz and K Fischer ldquoOpposing effects of heat stressonmale versus female reproductive success in Bicyclus anynanabutterfliesrdquo Journal of Thermal Biology vol 36 no 5 pp 283ndash287 2011

[31] G H Y Zhang Y Y Li K J Zhang J J Wang Y Q Liuand H Liu ldquoEffects of heat stress on copulation fecundityand longevity of newly-emerged adults of the predatory miteNeoseiulus barkeri (acari phytoseiidae)rdquo Systematic andAppliedAcarology vol 21 no 3 pp 295ndash306 2016

[32] M Tufail Y Nagaba A M Elgendy and M Takeda ldquoRegula-tion of vitellogenin genes in insectsrdquo Entomological Science vol17 no 3 pp 269ndash282 2014

[33] K-I Yano M T Sakurai S Izumi and S Tomino ldquoVitel-logenin gene of the silkworm Bombyx mori structure and sex-dependent expressionrdquo FEBS Letters vol 356 no 2-3 pp 207ndash211 1994

[34] S Zhang S Wang H Li and L Li ldquoVitellogenin a multivalentsensor and an antimicrobial effectorrdquo International Journal ofBiochemistry and Cell Biology vol 43 no 3 pp 303ndash305 2011

[35] N K Singh B C Pakkianathan M Kumar et al ldquoVitellogeninfrom the Silkworm Bombyx mori an effective anti-bacterialagentrdquo PLoS ONE vol 8 article e73005 2013

[36] M Fischer C Regitz M Kahl M Werthebach M Bolland U Wenzel ldquoPhytoestrogens genistein and daidzein affectimmunity in the nematode Caenorhabditis elegans via alter-ations of vitellogenin expressionrdquoMolecular Nutrition and FoodResearch vol 56 no 6 pp 957ndash965 2012

[37] K E Ihle O Rueppell Z Y Huang et al ldquoGenetic architectureof a hormonal response to gene knockdown in honey beesrdquoJournal of Heredity vol 106 no 2 pp 155ndash165 2015

[38] C M Fittinghoff and L M Riddiford ldquoHeat sensitivity andprotein synthesis during heat-shock in the tobacco hornworm

Manduca sextardquo Journal of Comparative Physiology B Biochem-ical Systemic and Environmental Physiology vol 160 no 4 pp349ndash356 1990

[39] B J Barber and N J Blake ldquoReproductive physiologyrdquo inScallops Biology Ecology and Aquaculture S E Shumway andG J Parsons Eds pp 357ndashe416 Elsevier Amsterdam TheNetherlands 2006

[40] P Sarup P Soslashrensen and V Loeschcke ldquoThe long-termeffects of a life-prolonging heat treatment on the Drosophilamelanogaster transcriptome suggest that heat shock proteinsextend lifespanrdquo Experimental Gerontology vol 50 pp 34ndash392014

[41] N E Gruntenko M Bownes J Terashima M Z Sukhanovaand I Y Raushenbach ldquoHeat stress affects oogenesis differentlyin wild-type Drosophila virilis and a mutant with altered juve-nile hormone and 20-hydroxyecdysone levelsrdquo Insect MolecularBiology vol 12 no 4 pp 393ndash404 2003

[42] J Xu J Shu X Qiu et al ldquoEffects of heat shock on ovarydevelopment and hsp83 expression in Tribolium castaneum(Coleoptera Tenebrionidae)rdquo Archives of Insect Biochemistryand Physiology vol 70 no 3 pp 204ndash216 2009

[43] X Cui F Wan M Xie and T Liu ldquoEffects of heat shock onsurvival and reproduction of two whitefly species Trialeurodesvaporariorum and Bemisia tabaci biotype Brdquo Journal of InsectScience vol 8 article 24 2008

[44] H FugoM Yamauchi and S G Dedos ldquoTesticular ecdysteroidlevel in the silkmoth Bombyx mori with special reference toheat treatment during the wandering stagerdquo Zoological Sciencevol 12 no 6 pp 783ndash788 1995

[45] L A F Teixeira and S Polavarapu ldquoHeat stress inhibits thecompletion of pupal diapause in Rhagoletis mendax (DipteraTephritidae)rdquo Annals of the Entomological Society of Americavol 98 no 2 pp 197ndash204 2005

[46] S-C Seehuus K Norberg U Gimsa T Krekling and G VAmdam ldquoReproductive protein protects functionally sterilehoney bee workers from oxidative stressrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 103 no 4 pp 962ndash967 2006

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


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International Journal of

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Zoology


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GenomicsInternational Journal of


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BioinformaticsAdvances in

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Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology

2 Psyche

heat insecticides and radiation becomes beneficial for manyorganism and life history traits [12ndash15] In Drosophila asignificant increase in lifespan of females repeatedly exposedto mild heat stress through hormesis has been suggestedby Hercus et al [16] In the oriental fruit moth Grapholitamolesta the exposure of females to a single heat event at 38∘Cfor 4 h caused an increase in the lifespan and length of theoviposition period leading to a potential increase in lifetimefecundity and suggesting hormesis [17] However Forbes [18]suggested that not all aspects of organism performance canbe hormetic simultaneously and there might be an energetictrade-off among life history traits Since both survival andreproduction require energy and since energy is limited itis expected that there will be a trade-off between these twofitness components [19]

In B mori although literatures are available on the effectof heat stress on survival and expression of heat shockproteins [8 20ndash25] little information is available on the effectof heat stress on its reproductive fitness It is suggested thatthough the increased expression of heat shock proteins undermild heat stress plays an important role in helping insect tosurvive its high level usually brings negative physiologicaleffects on organisms [26] The improved thermotoleranceat the cost of reduced reproductive performance has beenobserved inmany insect species such asBicyclus anynana andHelicoverpa armigera [27ndash30] In Spodoptera litura and Neo-seiulus barkerimild thermal stress increases thermotolerancebut brings a detrimental effect on fertility and fecundity[26 31]

The developing ovary in Bombyx consists of four poly-trophic meroistic ovarioles each of which contains a longlinear array of developing follicles in progressively advancingstage of development Each follicle is surrounded by a layerof follicle cells and contains an oocyte and interconnectednurse cells Vitellogenin a precursor of major egg yolkprotein plays an important role in promoting the growthand differentiation of developing oocytes The regulation ofvitellogenin is directly under the control of hormones atthe transcriptional level [32] In Bombyx its biosynthesisis regulated transcriptionally in a sex- and stage-dependentmanner [33] Vitellogenin functions not only as an energyreserve for the developing embryo but also in innate immuneresponse [34] Singh et al [35] showed the antibacterialactivity of vitellogenin and found that the infected silkwormlarvae treated with purified vitellogenin survived the full lifecycle in contrast to untreated animals Increased vitellogeninexpression in Caenorhabditis elegans potently increases theirresistance against pathogenic bacteria and heat [36] RecentlyIhle et al [37] showed that aging and lifespan in honeybees are affected by vitellogenin This protein influencesworker lifespan both as a regulator of behavioral matura-tion and through antioxidant and immune functions as anexperimental reduction of vitellogenin expression resulted indecreased lifespan and increased susceptibility to oxidativedamage

Since development time and reproductive output of aninsect are nearly as important to population growth as thesurvival of individuals the present study has been carriedout to investigate the consequences of heat stress on ovarian

development and fecundity as well as on vitellogenin geneexpression in B mori

2 Material and Methods

21 Insect Silkworm (Bombyx mori) L (CSR 18 a bivoltinerace) was used for present study Disease-free layings ofsilkworms breed CSR 18 were obtained from Central Ser-icultural Germplasm Resource Centre CSB Hosur TamilNadu and reared with fresh mulberry leaves in laboratoryunder controlled conditions (temperature 25plusmn1∘C humidity60ndash90 photoperiod 12L 12D) Larvae and pupae werestaged according to days after ecdysis and the availablemorphological markers such as spinneret pigmentation andgut purge

22 Heat Shock Treatment For heat shock treatment day 3spinning larvae and day 2 pupae were placed individually inglass test tubes plugged with cotton and were submerged in awater bath at 39∘C 42∘C and 45∘C for 1 h and 2 h respectivelyas described [38] After the treatment the larvae and pupaewere returned to controlled laboratory conditions Femalelarvae and pupae were considered for heat shock treatmentMale silkworm larvae and pupaewere subjected to heat shocktreatment only for the purpose of copulation

23 Determination of Gonadosomatic Index Heat shock wasgiven to day 2 female pupae and developing ovaries weredissected out after 48 h (day 4) 96 h (day 6) and 144 h(day 8) of heat shock treatment The isolated ovaries wererinsed with insect ringer solution blotted dry with filterpaper and the weight was measured using digital weighingbalanceGonadosomatic index (GSI)was calculated using theequation GSI = ovary wet weight (g)total body weight (g) times100 [39] The experiments were repeated 2-3 times and meanvalue of GSI was calculated for each experimental group fromthe data obtained from 6ndash8 individuals

24 Estimation of Fecundity Day 3 spinning larvae and day 2pupae (both males and females) were subjected to heat shocktreatment and the adult moths were collected for matingThe moths were allowed to copulate for 5 h and afterwardsthe moths were decoupled and the females were kept foroviposition and males were disposed off The fecundity wascalculated by counting the number of eggs laid by each femalewithin 24 h time period The experiments were repeated 3times and the mean value of fecundity was calculated fromthe data obtained from 10ndash12 individuals

25 RNA Isolation and Semiquantitative RT-PCR The fatbody and ovary were dissected out from 5th instar larvae andpupae under cold insect ringer solution and homogenizedimmediately in TRIzol reagent (Invitrogen) and stored atminus70∘C Total RNA was isolated according to the manufac-turerrsquos protocol The concentration of RNA was measuredspectrophotometrically at 260 nm The purity of the totalRNA was assessed using A

260280ratios Denaturing agarose

Psyche 3

C 39-1 39-2 42-1 42-2 45-1 45-200

05

10

15

20

25

30

35

NSNSNS

Day 4

NS

C 39-1 39-2 42-1 42-2 45-1 45-20

3

6

9

12

15

18

21

24NS

Day 6

NS

C 39-1 39-2 42-1 42-2 45-1 45-2

NS

Day 8

NS

Gon

ados

omat

ic in

dex

(times100

)

lowast

lowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

Gon

ados

omat

ic in

dex

(times100

)

Gon

ados

omat

ic in

dex

(times100

)

0369

1215182124273033363942





4 Psyche

C 39-1 39-2 42-1 42-20

100

200

300

400

500

600

NS NS NSNS

Num

ber o

f egg

s lai

dfe

mal

e


NS

lowastlowastlowast




3 Results





4 Discussion


Psyche 5

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12NS

NSNS NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowast

(a)C 39-1 39-2 42-1 42-2 45-1 45-2

00

04

08

12

16

20

24

28

32

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

-vg

-rp49

45-245-142-242-139-239-1C

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

(b)







6 Psyche

00

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

45-245-142-242-139-239-1C(a)

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(b)

C 39-1 39-2 42-1 42-2 45-1 45-200

02

04

06

08

10

12

14

NSNSNSNS NSNSNSNS

NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

Fat bodyOvary

NS

lowastlowastlowast

(c)





Psyche 7



Competing Interests


Acknowledgments


References























8 Psyche

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 3

C 39-1 39-2 42-1 42-2 45-1 45-200

05

10

15

20

25

30

35

NSNSNS

Day 4

NS

C 39-1 39-2 42-1 42-2 45-1 45-20

3

6

9

12

15

18

21

24NS

Day 6

NS

C 39-1 39-2 42-1 42-2 45-1 45-2

NS

Day 8

NS

Gon

ados

omat

ic in

dex

(times100

)

lowast

lowastlowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

Gon

ados

omat

ic in

dex

(times100

)

Gon

ados

omat

ic in

dex

(times100

)

0369

1215182124273033363942





4 Psyche

C 39-1 39-2 42-1 42-20

100

200

300

400

500

600

NS NS NSNS

Num

ber o

f egg

s lai

dfe

mal

e


NS

lowastlowastlowast




3 Results





4 Discussion


Psyche 5

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12NS

NSNS NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowast

(a)C 39-1 39-2 42-1 42-2 45-1 45-2

00

04

08

12

16

20

24

28

32

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

-vg

-rp49

45-245-142-242-139-239-1C

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

(b)







6 Psyche

00

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

45-245-142-242-139-239-1C(a)

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(b)

C 39-1 39-2 42-1 42-2 45-1 45-200

02

04

06

08

10

12

14

NSNSNSNS NSNSNSNS

NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

Fat bodyOvary

NS

lowastlowastlowast

(c)





Psyche 7



Competing Interests


Acknowledgments


References























8 Psyche

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 Psyche

C 39-1 39-2 42-1 42-20

100

200

300

400

500

600

NS NS NSNS

Num

ber o

f egg

s lai

dfe

mal

e


NS

lowastlowastlowast




3 Results





4 Discussion


Psyche 5

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12NS

NSNS NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowast

(a)C 39-1 39-2 42-1 42-2 45-1 45-2

00

04

08

12

16

20

24

28

32

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

-vg

-rp49

45-245-142-242-139-239-1C

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

(b)







6 Psyche

00

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

45-245-142-242-139-239-1C(a)

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(b)

C 39-1 39-2 42-1 42-2 45-1 45-200

02

04

06

08

10

12

14

NSNSNSNS NSNSNSNS

NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

Fat bodyOvary

NS

lowastlowastlowast

(c)





Psyche 7



Competing Interests


Acknowledgments


References























8 Psyche

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 5

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12NS

NSNS NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowast

(a)C 39-1 39-2 42-1 42-2 45-1 45-2

00

04

08

12

16

20

24

28

32

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

-vg

-rp49

45-245-142-242-139-239-1C

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

(b)







6 Psyche

00

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

45-245-142-242-139-239-1C(a)

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(b)

C 39-1 39-2 42-1 42-2 45-1 45-200

02

04

06

08

10

12

14

NSNSNSNS NSNSNSNS

NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

Fat bodyOvary

NS

lowastlowastlowast

(c)





Psyche 7



Competing Interests


Acknowledgments


References























8 Psyche

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 Psyche

00

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

45-245-142-242-139-239-1C(a)

C 39-1 39-2 42-1 42-2 45-1 45-200

04

08

12

16

20

24

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

NS

-vg

-rp49

45-245-142-242-139-239-1C

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowast

(b)

C 39-1 39-2 42-1 42-2 45-1 45-200

02

04

06

08

10

12

14

NSNSNSNS NSNSNSNS

NS

Relat

ive e

xpre

ssio

n of

vite

lloge

nin

mRN

A

Fat bodyOvary

NS

lowastlowastlowast

(c)





Psyche 7



Competing Interests


Acknowledgments


References























8 Psyche

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Psyche 7



Competing Interests


Acknowledgments


References























8 Psyche

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

8 Psyche

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article ovarian development and...

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