Reduced juvenile hormone synthesis in mosquitoes with low teneral reserves reduces ovarian previtellogenic development in Aedes aegypti

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Deirdre Flowers
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1 The Journl of Experimentl Biology 27, Pulishe y The Compny of Biologists 24 oi:1.1242/je Reue juvenile hormone synthesis in mosquitoes with low tenerl reserves reues ovrin previtellogeni evelopment in Aees egypti Arhim S. Croi 1, Yiping Li 2 n Fernno G. Norieg 2, * 1 Deprtmento e Prsitologi, Instituto e Cienis Biomeis, Universie e So Pulo, So Pulo, SP, CEP 5,58-9, Brzil n 2 Deprtment of Biologil Sienes, Flori Interntionl University, Mimi, FL 33199, USA *Author for orresponene (e-mil: noriegf@fiu.eu) Aepte 18 My 24 We investigte the reltionship mong nutritionl reserves, previtellogeni ovry evelopment n juvenile hormone (JH) synthesis in Aees egypti femle mosquitoes. By rising lrve uner ifferent nutritionl regimes, two ult phenotypes (lrge n smll femles) were generte, whih iffere signifintly in size t elosion (mesure y wing length). We mesure the totl mount of protein, lipis n glyogen in newly emerge (tenerl) lrge n smll femles. Tenerl reserves were signifintly lower in smll femles. Mximum previtellogeni ovry evelopment ourre only if enough tenerl nutrients were present. Mximum Summry previtellogeni ovry evelopment ws stimulte in smll femles with low tenerl nutrients y topilly pplying JH nlog. The iosyntheti tivity of Ae. egypti orpor llt (CA) ws stuie in vitro using riohemil metho. JH synthesis ws signifintly reue in femles emerge with low tenerl reserves n stimulte y sugr feeing. These results estlish tht the CA synthesizes enough JH to tivte ovry mturtion only in the presene of lrge nutrient reserves. Key wors: Aees egypti, mosquito, juvenile hormone, orpor llt, nutrition, ovry. Introution Oogenesis in mosquitoes is nutrient-limite proess, triggere only if suffiient reserves re ville (Briegel, 199; Wheeler, 1996). The primry folliles of Aees egypti re unifferentite t ult emergene n, uring the next 6 h, they grow to oule their size n reh the mximum previtellogeni length or resting stge (Hgeorn et l., 1977). This ovri previtellogeni phse of evelopment is initite only when nutrients re pproprite (Gwz n Spielmn, 1973). Femles must hve some mehnism to restrin ovry evelopment if nutrients re not ville, n hormonl ontrol system tivte y suitle nutritionl stimuli is very pproprite. Juvenile hormone (JH) is the key hormone regulting previtellogeni ovrin evelopment in mosquitoes (Hgeorn, 1994; Klowen, 1997). In newly emerge ult femle mosquitoes (tenerl femles), n inrese in JH signls tht eysis of the ult hs finishe n reproutive proesses shoul egin. JH levels in Ae. egypti inrese uring the first y fter ult emergene (Shpiro et l., 1986). This initil rise in JH is essentil for reproutive mturtion. JH ts on severl tissues, inluing ovries, ft oy n migut, mking them ompetent to perform their ult-speifi funtions (Klowen, 1997). JH is synthesize n relese from the orpor llt (CA), pir of enorine glns with nervous onnetions to the rin (Sty, 2). An inrese in rtes of JH iosynthesis is responsile for the rise in JH levels oserve uring the first y fter elosion (Li et l., 23). Is there reltionship etween the inrese of JH synthesis in the CA n the presene of nutrient reserves? In this stuy, we report tht low tenerl nutrient reserves reue JH iosynthesis n ovrin previtellogeni evelopment in Ae. egypti femles. Mterils n methos Chemils Juvenile hormone III (JH) ws from ICN (Irvine, CA, USA). The juvenile hormone nlog methoprene ws from Zoeon Co. (Plo Alto, CA, USA). Insets Aees egypti L. of the Rokefeller strin were rere t 28 C n 8% reltive humiity uner photoperio of 16 h:8 h light:rk. The lrvl iet ws 1% solution of ovine liver power (ICN, Auror, OH, USA). Five hunre lrve were rise in pns (23 m 35 m 13 m) ontining 1 liter of istille wter. Lrge or smll mosquitoes were otine y hnging the mount of iet e to the pn. Lrge mosquitoes were proue when lrve were rere in

2 2686 A. S. Croi, Y. Li n F. G. Norieg pns ing the following mount of iet: 1.5 ml on ys 1, 3, 4, 5 n 6 n.75 ml on y 2. Smll mosquitoes were proue when lrve were rere in pns with the following iet:.75 ml on ys 1, 3, 5 n 7. Uner these rering onitions, most lrve pupte t y 8. Virgin ult femles were offere otton wool p soke in wter or 15% surose solution. We refer to the otton wool p surose-fe femles s sugr-fe. Wing length n ovrin evelopment mesurements Ovries were isolte y tering the soft utile etween the fifth n sixth ominl sternites, pulling off n pling the terminl segments in rop of sline. Ovry n wing lengths were mesure uner isseting mirosope using n oulr mirometer. Wing length, mesure t ult emergene, esries the istne etween the point of rtiultion n the wing tip, exluing the fringe sles (Nsi, 199). Miroseprtion n nlysis of nutrients Miroseprtion of glyogen, lipis n proteins from the sme mosquito smples ws omplishe s esrie y Vn Hnel (1965) n moifie y Zhou et l. (24). Protein t were otine using the BCA protool (Piere, Rokfor, IL, USA). Glyogen ws mesure using the hot nthrone protool (Vn Hnel, 1985) n lipis using vnillin s esrie y Vn Hnel (1985). All iohemil nlyses were rrie out using triplite groups of five femles. In vitro riohemil ssy for CA tivity Preprtion of CA omplexes (CA tthe to the orpor ri n onnete to the rin) from ult Ae. egypti femles hs een previously esrie (Li et l., 23). Rtes of JH iosynthesis were estimte y the in vitro riohemil ssy, s esrie y Feyereisen n Toe (1981) n Feyereisen (1985) n moifie y Li et l. (23). The glns were inute in 1 µl M-199 ssy meium with lele L-[methyl- 3 H]methionine (speifi tivity TBq mmol 1 ; ~8 84 Ci mmol 1 ; Amershm Phrmi, Pistwy, NJ, USA). The finl onentrtion of methionine in the meium ws 5 µmol l 1 n the speifi tivity ws.56 TBq mmol l 1 (15 Ci mmol l 1 ). Uner these onitions, the inorportion of L-[methyl- 3 H]-methionine into JH ws liner for t lest 6 h (Li et l., 23). At the en of the experimentl perio, inutions were terminte y the ition of 1 µl 1% EDTA, n 1 µl methnol ontining µg of unlele JH s rrier n internl stnr. The inution meium n the gln were extrte together with 1 ml of hexne n seprte y thin-lyer hromtogrphy (TLC). After TLC seprtion [evelope in 2:1 (v/v) hexne:ethyl ette], the JH n ws etete uner UV light, ut, put into 1 ml sintilltion oktil overnight n ssye for 3 H. The quntities of JH proue in the experiment were lulte from the speifi tivity of the L- [methyl- 3 H]methionine in the meium with ssumption of speifi inorportion rtio of 1 (non-isotopi ilution). JH egrtion y esterses ws heke y inuting [ 3 H]JH in meium in the presene or sene of CA omplexes n nlyzing the reovery of lele JH % of the hormone ws reovere intt fter 4 h of inution (results not shown). In some experiments, the JH esterse inhiitor OTFP (Hmmok et l., 1984) ws e to the inution meium. Results of inutions in the presene or sene of OTFP were not signifintly ifferent. Sttistil nlysis Sttistil nlysis of the t ws performe y t-test or one-wy nlysis of vrine (ANOVA) with Tukey s post-test using GrphP Prism version 3. for Winows (GrphP Softwre, Sn Diego, CA, USA). The results were expresse s mens ± S.E.M. n were onsiere signifintly ifferent t P<.5. Results Effet of lrvl rering onitions on tenerl nutritionl reserves The two protools use for rering the lrve proue two istint tenerl ult phenotypes with signifint ifferenes in oy size. Ault femles emerge from lrve rise uner low iet protool (smll femles) were pproximtely.7% smller thn femles emerge from lrve rere uner high iet protool (lrge femles) [wing length: smll, 2.54±.5 mm (N=86) vs lrge, 3.42±.6 mm (N=84)]. Tenerl smll n lrge femles were nlyze within 2 h of elosion to etermine the totl mount of reserves present s lipis, glyogen n protein (Fig. 1). All three groups of nutrients were signifintly higher in tenerl lrge mosquitoes. Groups of smll n lrge femles fter elosion were kept on wter or sugr, n the totl mount of lipis, glyogen n protein ws mesure 24 n 48 h fter elosion (Fig. 2). Lipis n glyogen showe signifint inreses in oth smll n lrge sugr-fe femles. Effet of lrvl rering onitions on ult ovrin evelopment Fig. 3 shows the length of the terminl follile of ovries issete from wter-fe n sugr-fe smll n lrge femles. Folliles from newly emerge smll n lrge femles were ~4 µm long (Fig. 3A). 24 h fter emergene, the terminl folliles of lrge femles rise on wter or sugr h oule their size. The folliles of smll femles rise in wter h grown very little, while those of sugr-fe smll femles inrese their size y ~3% (Fig. 3B). Similr results were oserve t 48 h fter emergene; while folliles of smll femles rise on wter h grown to only 65 µm, those of sugr-fe smll femles inrese their size to ~83 µm ut were still signifintly smller thn the folliles from lrge femles (Fig. 3C). Effet of topilly pplie methoprene on ult ovrin evelopment To onfirm role for JH in previtellogeni ovry

3 Nutrients n juvenile hormone synthesis in mosquitoes 2687 evelopment, methoprene ( JH nlogue) ws topilly pplie to newly emerge smll mosquitoes (5 ng per.5 µl of etone). Aetone lone ws pplie to ontrols. After 24 h, the length of the terminl folliles from methoprene-trete smll femles ws signifintly lrger thn tht from smll femles tht were topilly pplie with etone (Fig. 4). Topilly pplie methoprene inue the evelopment of previtellogeni ovries in smll mosquitoes to levels omprle with those oserve in lrge sugr-fe femles. JH synthesis in smll n lrge femles The sl rte of JH iosynthesis y CA issete from smll n lrge mosquitoes ws mesure t ifferent times fter emergene using the in vitro riohemil ssy. The iosyntheti tivity of the Ae. egypti CA ws signifintly reue in femles emerge with low tenerl reserves Lipis (µg/femle) Glyogen (µg/femle) Protein (µg/femle) Lipis Glyogen Protein Fig. 1. Nutritionl reserves in newly emerge femles. Nutrients from tenerl femles were nlyze within 2 h of elosion., smll tenerl mosquito;, lrge tenerl mosquito. Eh r represents the men ± S.E.M. of three inepenent etermintions of groups of five mosquitoes. Asterisks enote signifint ifferenes in eh group (unpire t-test; P<.1). (Fig. 5A). Rising the smll femles on high sugr iet (15%) resulte in signifint inrese of JH synthesis ompre with smll femles rise on wter (Fig. 5B). Disussion Differenes in the mount of reserves umulte uring the lrvl life ffet egg mturtion in mosquitoes (Briegel, 199). While mosquitoes rere in the lortory n e provie with n optiml iet, those in the fiel often enounter suoptiml onitions tht result in high vriility in tenerl Lipis (µg/femle) Glyogen (µg/femle) Protein (µg/femle) SW SS 15% LW LS 15% Fig. 2. Nutritionl reserves in femles fe on sugr. Ault femles were offere otton wool p soke in either wter or 15% surose solution. Nutrients were nlyze t 24 or 48 h fter elosion. SW, smll femles rise on wter; SS, smll femles rise on sugr; LW, lrge femles rise on wter; LS, lrge femles rise on sugr. Eh r represents the men ± S.E.M. of three inepenent etermintions of groups of five mosquitoes. Vlues lele with ifferent letters re signifintly ifferent y Tukey s test fter ANOVA t P< Lipis Glyogen Protein Hours fter elosion

4 2688 A. S. Croi, Y. Li n F. G. Norieg Follile length (µm) A B C Fig. 3. Effet of nutrients on previtellogeni follile evelopment. We mesure the length of the terminl follile of ovries issete from wter-fe n sugr-fe smll n lrge femles. (A) Folliles from newly emerge femles., smll tenerl mosquito;, lrge tenerl mosquito. (B) Folliles from femles 24 h fter elosion. (C) Folliles from femles 48 h fter elosion. SW, smll femles rise on wter; SS, smll femles rise on 15% sugr; LW, lrge femles rise on wter; LS, lrge femles rise on 15% sugr. Eh r represents the men ± S.E.M. of t lest 2 inepenent etermintions of iniviul femles. Vlues lele with ifferent letters re signifintly ifferent y Tukey s test fter ANOVA t P<.5. size n reproutive potentil (Nsi, 199; Feinso n Spielmn, 198; Tun-Lin et l., 2). Rering onitions n e mnipulte in the lortory to eluite the effets of suoptiml environmentl onitions. We generte two very istint ult phenotypes (lrge n smll femles) y rising lrve uner ifferent iets. Femles tht were signifintly smller in size t elosion (mesure y wing length) h signifintly lower lipi, protein n glyogen tenerl reserves thn lrger femles. Our stuies onfirm similr onlusions from omprehensive reports previously pulishe (Briegel, 199; Briegel et l., 21). Newly emerge femles utilize SW SS LW LS Newly emerge 24 h 48 h SW SS LW LS Follile length (µm) SW SW+JH SS SS+JH LS Fig. 4. Effet of juvenile hormone (JH) on previtellogeni follile evelopment. Follile length ws mesure 24 h fter elosion. SW, smll femles rise on wter; SS, smll femles rise on sugr; SS+JH, smll femles rise on sugr n trete with methoprene; SW+JH, smll femles rise on wter n trete with methoprene; LS, lrge femles rise on sugr; JH, femles trete with methoprene (5 ng per.5 µl etone) within 1 h of elosion. Controls (SW, SS n LS) were trete with.5 µl of etone. Eh r represents the men ± S.E.M. of 1 inepenent etermintions of iniviul femles. Vlues lele with ifferent letters re signifintly ifferent y Tukey s test fter ANOVA t P<.5 or higher. reserves rrie over from the pupl stge ut soon seek out sugr mels s n importnt energy soure (Clements, 1992). High sugr mels le to n inrese in glyogen n lipi reserves. Growth of the primry follile to the resting stge in Ae. egypti is liner n rehes mximum evelopment out 6 h fter ult elosion (Hgeorn et l., 1977). Folliles of smll mosquitoes i not evelop muh eyon the tenerl stge when they were kept on wter for 48 h. By ontrst, sugr feeing permitte prtil growth of ovrin folliles in smll mosquitoes. Feinso n Spielmn (198) lso reporte tht surose mel stimultes prtil evelopment of folliles from nutrient-eprive ult Ae. egypti. There re severl reports esriing orreltions etween tenerl nutritionl reserves, JH tivity n previtellogeni egg mturtion in nutogenous mosquitoes (Le, 1963; Gwz n Spielmn, 1973; Hgeorn et l., 1977; Feinso n Spielmn, 198). Depittions, CA removl n ominl ligtions were use to prove tht the growth of the previtellogeni folliles is uner the ontrol of ftors from the rin n CA (Le, 1963; Gwz n Spielmn, 1973; Hgeorn et l., 1977). Depittion within 1 h of emergene or CA removl soon fter elosion prevents ovrin previtellogeni growth. Topil pplition of JH nlogs stimultes norml growth of previtellogeni ovries in epitte or CA-lte tenerl femles (Gwz n Spielmn, 1973; Hgeorn et l., 1977). We onfirme the role of JH on ovry evelopment. A topilly pplie JH nlogue stimulte previtellogeni ovrin evelopment in nutrienteprive mosquitoes. Folliles of methoprene-trete smll femles ttine norml previtellogeni growth.,

5 Nutrients n juvenile hormone synthesis in mosquitoes 2689 JH synthesis (fmol CA 1 h 1 ) A B Lrge mosquito Smll mosquito Smll wter JH levels in Ae. egypti re low t elosion, inrese uring the first y fter ult emergene n remin high in sugrfe femles (Shpiro et l., 1986). This initil rise in JH is essentil for femle s reproutive mturtion (Klowen, 1997). Rtes of JH iosynthesis y the CA in vitro losely reflet the levels of JH in the mosquito; iosynthesis of JH is very low in newly emerge femles n inreses rmtilly uring the first 24 h fter ult elosion (Li et l., 23). We hve investigte for the first time the effet of lrvl iet on synthesis of JH y the CA of tenerl femles. We oserve tht iosynthesis of JH y the Ae. egypti CA ws signifintly reue in femles emerge with low tenerl reserves (smll femles). When smll femles were fe on high sugr iet, JH synthesis ws signifintly inrese. How o nutrients tivte JH synthesis fter ult elosion? Alltotropins (AT) re pepties tht stimulte JH synthesis y the CA (Ktok et l., 1989; Veenstr n Costes, 1999). We hve previously esrie tht the CA of newly emerge mosquito nees to e expose to Ae. egypti AT efore it is ple of synthesizing JH (Li et l., 23). Stuies using ntioies ginst Ae. egypti AT showe tht the peptie is present in ells of the rin of Aees n Anopheles mosquitoes (S. Hernánez-Mrtínez n F.G.N., * Smll sugr ** Hours fter elosion Fig. 5. Effet of nutrients on iosynthesis of juvenile hormone (JH) in vitro. JH synthesis ws evlute in vitro in CA omplexes issete t ifferent times fter emergene. (A) Synthesis in lrge n smll mosquitoes rise on wter. (B) Smll mosquitoes with low tenerl reserves were rise on wter or sugr. Eh r represents the men ± S.E.M. of five inepenent etermintions of iniviul orpor llt (CA) omplexes. Asterisks enote signifint ifferenes in eh group (unpire t-test; *P<.5, **P<.5 n P<.1). unpulishe oservtions). Removl of the meil neuroseretory ells (mn) from Ae. egypti immeitely fter ult emergene suppresses egg mturtion; in oler femles, mn ltion hs little effet (Le, 1967). It is resonle to hypothesize tht AT is one of the ftors from the he tht is essentil for reproutive mturtion. When the mount of nutrients is pproprite, the rin woul relese AT, n the CA woul eome ple of synthesizing enough JH to tivte reproutive mturtion. Therefore, the previtellogeni mturtion of ovries seems to epen exlusively on the pity of the CA to proue high mounts of JH uring the first y fter the imginl molt. A similr mehnism ontrols ipuse in mosquitoes. Reio et l. (1999) reporte tht ipuse in the mosquito Culex pipiens is use y inhiition of JH synthesis y the CA. Nonipusing ult femles synthesize four times more JH thn o ipusing mosquitoes. Although smll quntities of JH re proue uring ipuse, ipusing femles lk suffiient JH to stimulte growth of ovrin folliles to the resting stge. How re nutrients sense? A reent pper y Colomni et l. (23) esries nutrient sensor mehnism tht ontrols Drosophil growth. These uthors propose moel where the mino-i-responsive serine/threonine protein kinse trget of rpmyin (TOR) signling in the ft oy moultes insulin signling n nutrient-epenent responses in peripherl tissues. The rin mn sense high irulting rohyrte levels n serete insulin-like pepties (ILPs), whih intert with ftors relese y the ft oy in response to mino is n inrese insulin signling in the peripherl tissues. Aoring to this moel, the sensing of mino is in the ft oy n the sensing of rohyrtes in the rin re importnt for the regultion of nutrient-epenent responses. One of the most iffiult hllenges in the future will e to isover whether one type of nutrient is more importnt thn nother in triggering JH seretion, s well s the mehnisms y whih mosquitoes sense n respon to hnges in their nutritionl reserves. In summry, CA tivity is promote y the presene of enough nutrient stores; inequte lrvl nutrition prevente the erly pek of JH iosynthesis tht we etete in lrge femles. There seems to e mehnism involving minimum threshol of nutrients tht eliits the relese of AT y the rin. We thnk Roin K. Rohe for inset re, Phil Evns for the gift of methoprene n Mihel R. Roe for the OTPF gift. This work ws supporte y NIH grnt AI to F.G.N. Referenes Briegel, H. (199). Metoli reltionship etween femle oy size, reserves, n feunity of Aees egypti. J. Inset. Physiol. 36, Briegel, H., Wltert, A. n Kuhn, R. (21). Reproutive physiology of Aees (Aeimorphus) vexns (Dipter: Culiie) in reltion to flight potentil. J. Me. Entomol. 38, Clements, A. N. (1992). The Biology of Mosquitoes. Lonon: Chpmn n Hll. Colomni, J., Risin, S., Pntli, S., Rimerski, T., Montgne, J. n

6 269 A. S. Croi, Y. Li n F. G. Norieg Leopolo, P. (23). A nutrient sensor mehnism ontrols Drosophil growth. Cell 114, Feinso, F. M. n Spielmn, A. (198). Nutrient-meite juvenile hormone seretion in mosquitoes. J. Inset Physiol. 26, Feyereisen, R. (1985). Riohemil ssy for juvenile hormone III iosynthesis in vitro. In Methos in Enzymology, vol. III (e. J. H. Lw n H. C. Rilling), pp Orlno, FL: Aemi Press. Feyereisen, R. n Toe, S. S. (1981). A rpi prtition ssy for routine nlysis of JH relese y inset CA. Anl. Biohem. 111, Gwz, R. W. n Spielmn, A. (1973). Corpus lltum ontrol of ovrin evelopment in Aees egypti. J. Inset Physiol. 19, Hgeorn, H. H. (1994). The enorinology of the ult femle mosquito. Av. Dis. Vetor. Res. 1, Hgeorn, H. H., Turner, S., Hgeorn, E. A., Ponteorvo, D., Greenun, P., Pfeiffer, D., Wheelok, G. n Flngn, T. R. (1977). Post emergene growth of the ovrin folliles of Aees egypti. J. Inset Physiol. 23, Hmmok, B. D., Ael-Ael, Y. A. I., Mullin, C. A., Hnzlik, T. N. n Roe, R. M. (1984). Sustitute thiotrifluoropropnones s potent seletive inhiitors of juvenile hormone esterse. Pestiie Biohem. Physiol. 22, Ktok, H., Toshi, A., Li, J. P., Crney, R. L., Shooley, D. A. n Krmer, S. J. (1989). Ientifition of n lltotropin from ult Mnu sext. Siene 243, Klowen, M. J. (1997). Enorine spets of mosquito reproution. Arh. Inset Biohem. Physiol. 35, Le, A. O. (1963). Some reltionships etween environment, orpor llt, n egg mturtion in Aeine mosquitoes. J. Inset Physiol. 9, Le, A. O. (1967). The meil neuroseretory ells n egg mturtion in mosquitoes. J. Inset Physiol. 13, Li, Y., Hernánez-Mrtínez, S., Unnithn, G. C., Feyereisen, R. n Norieg, F. G. (23). Ativity of the orpor llt of ult femle Aees egypti: effets of mting n feeing. Inset Biohem. Mol. Biol. 33, Li, Y., Unnithn, G. C., Veenstr, J. A., Feyereisen, R. n Norieg, F. G. (23). Stimultion of JH iosynthesis y the orpor llt of ult femle Aees egypti in vitro: effet of frnesoi i n Aees lltotropin. J. Exp. Biol. 26, Nsi, R. S. (199). Reltionship of wing length to ult ry weight in severl mosquito speies (Dipter: Culiie). J. Me. Entomol. 27, Reio, J., Chen, M. H. n Meol, R. (1999). Juvenile hormone iosynthesis in ipusing n nonipusing Culex pipiens (Dipter: Culiie). J. Me. Entomol. 36, Shpiro, A. B., Wheelok, G. D., Hgeorn, H. H., Bker, F. C., Tsi, L. W. n Shooley, D. A. (1986). Juvenile hormone n juvenile hormone esterse in ult femles of the mosquito Aees egypti. J. Inset Physiol. 32, Sty, B. (2). A review of the role of neuroseretion in the ontrol of juvenile hormone synthesis: triute to Bert Shrrer. Inset Biohem. Mol. Biol. 3, Tun-Lin, W., Burkot, T. R. n Ky, B. H. (2). Effets of temperture n lrvl iet on evelopment rtes n survivl of the engue vetor Aees egypti in north Queensln, Austrli. Me. Vet. Entomol. 14, Vn Hnel, E. (1965). Miro seprtion of glyogen, sugrs n lipis. Anl. Biohem. 11, Vn Hnel, E. (1985). Rpi etermintion of glyogen n sugrs in mosquitoes. J. Am. Mosq. Contr. Asso. 1, Vn Hnel, E. (1985). Rpi etermintion of totl lipis in mosquitoes. J. Am. Mosq. Contr. Asso. 1, Veenstr, J. A. n Costes, L. (1999). Isoltion n ientifition of peptie n its DNA from the mosquito Aees egypti relte to Mnu sext lltotropin. Pepties 2, Wheeler, D. (1996). The role of nourishment in oogenesis. Annu. Rev. Entomol. 41, Zhou, G., Flowers, M., Frierih, K., Horton, J., Pennington, J. n Wells, M. A. (24). Metoli fte of [ 14 C]-lele mel protein mino is in Aees egypti mosquitoes. J. Inset Physiol. 5,

EFFECT OF SOYBEAN CYST NEMATODE ON GROWTH OF DRY BEAN. Research Report to Northarvest Bean Growers, January 19, 2009

EFFECT OF SOYBEAN CYST NEMATODE ON GROWTH OF DRY BEAN Reserh Report to Northrvest Ben Growers, Jnury 19, 29 Berlin D. Nelson, Susilo Poromrto, n Ruell Goswmi, Dept. Plnt Pthology, NDSU Ojetive: Determine