OXYGEN REQUIREMENTS AND THE PHYSIOLOGICAL SUPPRESSION OF SUPERNUMERARY INSECT PARASITOIDS

p. Biol. (96), 0, 550 textfigures Printed in Gret Britin OXYGEN REQUIREMENTS AND THE PHYSIOLOGICAL SUPPRESSION OF SUPERNUMERARY INSECT PARASITOIDS BY RODERICK C. FISHER Deprtment of Zoology nd Comprtive University College London Antomy, (Received 6 April 96) INTRODUCTION There re mny references, in the literture of biologicl control, to the physiologicl suppression of competing endoprsitic Hymenopter. As phenomenon it hs received vriety of explntions, such s the secretion of some toxic substnce by the surviving prsitoid (Timberlke, 90, 92), or of cytolytic enzyme which destroys its competitors (Spencer, 926; Thompson & Prker, 90). None of these explntions, however, hs been ccompnied by ny criticl exmintion of its possible vlidity. In n erlier pper of this series (Fisher, 966) the vilbility of oxygen to the host insect ws shown to hve n importnt effect on the outcome of competition between its prsitoids. The norml outcome of such competition is for the younger one to be suppressed nd for the older to survive. This cn be reversed by rising the oxygen content of the ir surrounding the host to % or 50% by volume. Conversely, the effects of lowering the oxygen content of the tmosphere by the ddition of nitrogen is to retrd the development of both prsites, prticulrly the younger one. The development of single eggs or of young lrve is rrested nd lter they become invested with sphericl cpsule of blood phgocytes in exctly the sme mnner s do younger lrve when suppressed in the presence of n older one. Internl prsitoid lrve of the Ichneumonide mke no direct contct with the trchel system of their hosts nd obtin their oxygen directly from the host hemolymph in which they lie. There is no respirtory pigment nd the hemolymph contins oxygen in simple solution in concentrtion which is proportionl to the prtil pressure of oxygen in the tmosphere surrounding the cterpillr, nd depends lso upon the osmotic pressure of the hemolymph. It ws therefore postulted tht lck of oxygen in the host hemolymph due to the respirtory ctivity of the older prsite lrv ws responsible for the physiologicl suppression of the younger one. In order to test the vlidity of this hypothesis the reltionship between oxygen tension of the tmosphere surrounding the host nd tht in its hemolymph hs been studied, both before prsitiztion nd during the course of prsite development. Secondly, the effects of vrying oxygen tension in the host hemolymph on the behviour nd survivl of the prsite lrve hve been ssessed during the period of erly development when suppression normlly occurs. Thirdly, the oxygen uptke of the host nd prsite hve been mesured for the sme period. Exp. Biol. 0,

52 RODERICK C. FISHER MATERIAL AND METHODS The hymenopterous prsitoids used in this work re the ichneumon wsps Nemeritis ccmescens Grv. nd Horogenes chrysostictos Gmelin (Ophionine). Both species ttck mture lrve of the moth Ephesti kuhmeu Hb. (=sericrhtm Scott) (Phycitide). All three species re mintined in lbortory culture by methods lredy described (Fisher 96 ). Since it hs lredy been shown tht the mechnism of physiologicl suppression is the sme in both intr nd interspecific competition between these two prsitoids (Fisher 966) most of the work described here ws crried out with Nemeritis, prthenogenetic species which cn be bred more esily nd more bundntly in the lbortory thn Horogenes. Two methods for mesuring the oxygen content of Ephesti blood were used. Roughton & Scholnder's (9) method relies on the extrction of dissolved gses from 0/tl. blood smple. Since mture Ephesti cterpillr hs only bout 0 /il. of blood, pooled smples from five or more cterpillrs were required for ech determintion. Becuse of the difficulty of collecting relible smples of sufficient volume for estimtion by this method, nd lso becuse the oxygen content of the blood proved to be of the sme order s tht lredy in solution in the regents used in the process of extrction, the ccurcy of the results could not be relied upon. Consequently the oxygen estimtion method of Krogh (9) s modified bypryor (95 5) ws dopted in lter work nd used for the mjority of results recorded in this pper. In this technique smll bubble of ir or known gs mixture of oxygen nd nitrogen is injected into the insect's body cvity. The gses in the bubble re llowed to equilibrte with those in solution in the blood for severl hours before the insect is cut open under glycerine. The bubble is then removed in glycerinefilled pipette to bridge slide contining glycerine. The dimeter of the bubble is then mesured microscopiclly before nd fter the bsorption of its contined oxygen with lkline pyrogllol. The volume chnge cn then redily be clculted. The method is only ccurte within bout 2% but it gives quick indiction of the percentge sturtion with oxygen in individul insects. Controls were run with ech set of redings, using either ir or oxygennitrogen mixtures, bubbled into glycerine. The respirtion of Nemeritis lrve in the first, second nd third instrs ws mesured using the Crtesin diver respirometer (Holter, 9). The prsitoid lrve were obtined by dissecting the host cterpillrs in insect Ringer; they were then trnsferred to the bulb of the diver in smll drop of Ringer, the neck sels were plced in position nd the oxygen uptke ws mesured over period of 2 hr. t 20 0 C. The oxygen consumption of helthy nd prsitized Ephesti lrve ws mesured with the use of stndrd Wrburg respirometer. Atmospheres for the study of prsite survivl under vried conditions of oxygen tension were obtined by filling n spirtor of 2. cpcity with vrious mixtures of oxygen nd nitrogen. The gs mixture ws humidified nd equilibrted t the incubtor temperture of 25 0 C. by being bubbled through wsh bottle of wter before being pssed, t rte of 05. per hour, through 6 x in. glss tubes contining the prsitized lrve. Gs exit from the tubes ws provided by cpillry vlves.

Physiologicl suppression of supernumerry insect prsitoids 5 EXPERIMENTS AND RESULTS Oxygen content of Ephesti blood Estimtion of the dissolved oxygen content of Ephesti lrvl hemolymph by the Roughton & Scholnder (9) method gve men vlue 09 + 055 (^ = A 2 ) v l O 2 per 00 vol. hemolymph, nd by the modified Krogh's method (Pryor, 955) the hemolymph ws found to be in equilibrium with n tmosphere contining *0 ±i*2% oxygen by volume. This is equivlent to clculted solubility of 07 vol. O 2 %. By using the ltter method the oxygen content of gs bubbles in equilibrium with the hemolymph of Ephesti cterpillrs mintined in vrious oxygen/nitrogen W s / i V XI O M r bo c o C s V Q. mph X. o E rt f~ S E JO o V c 0 20 _ 0 / / // '/ \ / A \ / * / / * y \ y Ki 0 20 0 Percentge O 2 In tmosphere i t i 0 50 Fig.. Reltionship between the oxygen tension of the tmosphere nd tht of gs bubbles equilibrted with the hemolymph of Ephtx lrve. Ech point represents the men of twenty redings together with their stndrd devition. Tble. The solubility of oxygen in hemolymph of Ephesti % oxygen in tmosphere 5 0 i % oxygen in gs bubble equilibrted with hemolymph 22 26 Solubility of O t in i6%nclt2o C. in ml./i. 75 275 56 88 065 5 No oxygen detected. Clculted solubility of O, in hemolymph t 20 0 C. in ml./l. 0825 7 586 692 88 mixtures, of rnge 250 % oxygen, ws found to increse with the prtil pressure of oxygen in the tmosphere surrounding the cterpillr (Fig. ). Thus it is possible to use the prtil pressure of oxygen in the tmosphere surrounding the host to lter the 2

5 RODERICK C. FISHER oxygen tension in the host hemolymph. Furthermore, the ctul oxygen content IP the hemolymph t ny given oxygen tension my be clculted from the freezingpoint depression of 5 C. mesured by Rouschl (90) for the closely relted species E. eluteu, which is equivlent to i*6% solution of sodium chloride (Tble ) nd the known solubility of oxygen (Prosser, 950). The reltionship between prsite development nd the oxygen content of the host hemolymph ws studied by mking series of estimtions t ech lrvl instr of the prsite (Fig. 2). It ws found tht the oxygen content of the bubbles equilibrted with the host hemolymph flls with the progress of prsitism from the norml level of % to men minimum of 5 % when the contined prsite lrv reched the fifth nd finl lrvl instr. This stge of the prsitoid is polypneustic nd breks out of the host skin soon fter moulting from the fourth instr. 6. = 2 * E I 0 6 i 8 6 ( R, Unprsitlzed \jfc»t Instr \ 2nd Inttr rdinitr ' \ N th \ 0 2 S 6 7 8 Length of contined Nemeritis lrv (mm.) \ instr,5th liutr Fig. 2. Reduction in the oxygen tension in gs bubbles equilibrted with Ephesti hemolymph due to prsitism by Nemeritis. Hosts kept in norml ir t 25 0 C.; men of twenty observtions t ech point with stndrd devition*. Oxygen consumption of Nemeritis lrve The oxygen uptke of young Nemeritis lrve in the first, second nd third instrs were mde with the Crtesin diver respirometer for comprison with the oxygen vilbility in the host hemolymph. The results (Fig. ) show the rte of oxygen consumption in microlitres per hour t N.T.P. plotted ginst the body length of the individul prsite mesured with n eyepiece micrometer. The minimum uptke mesured ws bout O'Ooo6 /xl./hr. for young firstinstr lrve, but this rose rpidly with ge, incresing bout eightfold by the end of the first instr.

Physiologicl suppression of supernumerry insect prsitoids 55 Oxygen consumption of Ephesti cterpillrs The oxygen uptke of both helthy nd prsitized Ephesti lrve of 25 mg. live weight ws mesured, primrily to give some indiction of its rte in comprison with those of its prsites. However, since the respirtory rte vried with the ctivity of the cterpillr, some form of continuously recording respirometer would be necessry to give complete picture of the host respirtion during the course of prsite development. Nevertheless, the results obtined with the Wrburg pprtus (Fig. ) show tht there is grdul fll in the rte of oxygen consumption of the prsitized Ephesti, prticulrly s the contined prsite pproches mturity nd the host becomes more nd more lethrgic. The oxygen consumption of helthy, unprsitized Ephesti is shown for comprison. 0 Egg st instr c 0 02 00 I 0 5 20 25 Length of prsite (mm.) Fig.. The oxygen uptke of Nemeritis lrve in reltion to body length in thefirstnd second instrs. The rrow shows the point t which the prsite first becomes cpble of suppressing its younger competitors. Survivl of Nemeritis in reduced oxygen tensions Since the host hemolymph hs cpcity for oxygen which is dependent upon the prtil pressure of oxygen in the insect's trchee nd the osmotic pressure of the hemolymph, ny increse in the oxygen demnds of the prsite due to its growth must bsorb ll the dissolved oxygen vilble in the hemolymph of the host nd possibly produce n increse in the oxygen uptke into the hemolymph from the host's trche. This follows from the observtion tht the oxygen consumption of newly htched lrve of Nemeritis (00006 pl.[hi.) is roughly equl to the totl oxygen ^pntent of the hosts' hemolymph t ny given time (005 pi. per cterpillr of 0 pi.

i 56 RODERICK C. FISHER totl hemolymph volume). Becuse of the mny difficulties which rise in ttempting to culture internl prsitoids in rtificil medi outside their hosts, the effects on the prsitoid of lowering the oxygen tension of the host hemolymph hve been observed in vivo. This ws done by ltering the prtil pressure of oxygen in the tmosphere surrounding the host, since it hs been shown tht the oxygen content of the hemolymph vries directly with the prtil pressure of oxygen in the tmosphere (Fig. ). Experimentl stocks of 00 singly prsitized Ephesti were kept t ech prtil pressure of oxygen. Ten cterpillrs were removed nd dissected ech dy from ech stock for the first 0 dys of their development in order to exmine the condition of their contined prsites. Becuse of the necessity for dissection of the hosts the survivl of individul prsites could not be followed. Helthy Prsitized by NuntrHIt 8 E««st instr 2nd rd th 5th r 6» I 2 0 08 I. 06 ** «.! <M 02 I I 0 2 5 6 Length of prsite (mm.) Fig.. Grph showing the oxygen uptke of Ephesti cterpillrs in reltion to prsitism by Nemeritis. Humid gs mixtures contining either 2, 5, or 0% oxygen in different tests were pssed through glss tubes contining the lrve. Growth curves obtined for Nemeritis t different oxygen tensions re compred with those obtined in norml ir in Fig. 5. The lowering of the oxygen tension of the host hemolymph by this mens ffects the rte of prsite development, primrily by delying the htching of the egg, but retrdtion of development in the first instr lso occurs. The condition of the prsitoids, prticulrly those rered in 5 nd 2 % oxygen, closely resembles tht of physiologiclly suppressed lrve in cses of multiprsitism. Retrded firstinstr lrve remin very smll, curled up upon themselves nd lmost motionless. When they become moribund they re ttcked by the host hemocytes in mnner exctly similr to tht seen in cses of multiprsitic physiologicl suppression. Furthermore, there is fr greter mortlity of eggs nd young firstinstr lrve in 2 nd 5 % O thn t 0 nd 2 % (ir controls). Mortlity of

Physiologicl suppression of supernumerry insect prsitoids 57 prsites in ech dy's smple is given in Tble 2 nd the totl from ech line represents the percentge mortlity of 00 prsites observed in the 0dy period. In ddition, it my be noted tht the mortlity due to oxygen lck in these test is greter in the first 6 dys thn subsequently; from which it my be supposed tht the lte firstinstr lrv nd those in subsequent instrs re better ble to survive low oxygen tension thn the eggs nd newly htched lrve. The differentil survivl of the instrs with respect to very low oxygen tension ws exmined further by keeping Nemeritis eggs nd lrve of ll instrs individully in glss cells of pproximtely 00 mm, cpcity, completely filled with liquid prffin nd seled, without including ir bubbles, with gresed coverslip. The ctul volume of oxygen dissolved in the prffin ws not mesured, but in qulittive test, drops 0 2 Fig. 5. The effect of lowering the tmo»pheric oxygen tension on the growth rte of Nemeritis eggs nd lrve t 5 C. Tble 2. The mortlity of Nemeritis lrve during the first 0 dys of development under conditions of reduced oxygen tension, t 25 0 C. Totl Dy 8 0, mortlity) i % O, (controls) 0% O, 5%O, % O, 8 9 0 0 68 6 9 of lkline pyrogllol injected nerobiclly into the cells did not drken t ll rpidly, though they eventully turned ple strw colour fter period of 8 dys. Since the prsite's spircles remin closed until the finl instr is reched the closed respirtory system of the lrv is not impired by immersion in liquid prffin. Eggs nd lrve 8 5 8 9 6 9 6 0 ' 88

58 RODERICK C. FISHER in the first to fourth instrs were immersed individully in the glss cells nd observed" dily during incubtion t 25 C. The eggs showed no development fter 2 hr. nd by the sme time ll the newly htched firstinstr lrve (less thn io mm. long) hd lso died. Lte firstinstr lrve nd those in subsequent instrs remined live nd were moving normlly dys lter. They continued to 'feed* so tht by the fourth dy their midguts were distended with globules of prffin. One of the thirdinstr lrve hd successfully moulted to the fourth instr. An pneustic fourth instr hd lso by this time moulted to the fifth instr whose spircles re normlly open for tmospheric respirtion fter it leves the host. Liquid prffin hd entered the trchel system nd the lrv ws ded. Evidently the eggs nd newly htched lrve hve low, but bsolute, requirement for oxygen which must be stisfied if they re to survive, while mture firstinstr lrve nd those in subsequent instrs, with the exception of the lst, re pprently ble to survive nd feed in very low oxygen tensions. DISCUSSION In n erlier pper (Fisher, 96 b) the vilbility of oxygen to competing prsitoids ws shown to be importnt in controlling the outcome of multiprsitic competition. An hypothesis of sphyxition ws put forwrd s possible explntion of the physiologicl suppression of supernumerry insect prsitoids. The experiments described here were crried out to scertin whether the respirtory ctivity of the relevnt stges of the prsite lrve nd the oxygen content of the host hemolymph would support this hypothesis. The oxygen content of the hemolymph of unprsitized Ephesti cterpillrs kept in ir, though low compred with blood tht contins respirtory pigment, is in fct bout 66 % sturted with oxygen nd pproches the vlue for the solution of oxygen in n equivlent slt solution (0*56 vol. % for i6 solution of sodium chloride). It is pprent tht the hemolymph of Ephesti crries no more oxygen thn physicl solution will permit. This is in ccordnce with other determintions mde on the hemolymph of lepidopterous lrve (Adler, 97; Bbers, 98; Pryor, 955). Mesurement of oxygen tensions in the hemolymph over rnge of prtil pressures of oxygen show tht, in tmospheres contining from ioto 50% of O s byvol. the dissolved oxygen content vries in direct proportion to its prtil pressure in the tmosphere surrounding the insect. Thus, by keeping the hosts in vrious oxygen/ nitrogen mixtures, it ws possible to exmine the effects of oxygen tension on the behviour nd survivl of the prsite. When this ws done it becme immeditely pprent tht lowering the oxygen tension of the hemolymph cuses considerble dely in the time tken for the egg to htch nd lso retrds the subsequent lrvl development. In cses of extreme retrdtion, prticulrly in 2 % oxygen, mny of the eggs nd lrve become surrounded by the host's hemocytes s in true physiologicl suppression. It ws noticeble, however, tht the retrding effects of reduced oxygen tension decrese with the incresing ge of the prsite lrv, especilly fter the first instr is completed. This differentil tolernce ws confirmed by testing the survivl of ll

Physiologicl suppression of supernumerry insect prsitoids 59 instrs in seled glss cells of liquid prffin in which the oxygen tension is very low. Eggs nd newly htched lrve cnnot survive in liquid prffin, wheres the first nd subsequent instrs cn do so. Evidently the former hve very low, but bsolute, requirement for oxygen which must be stisfied by the host if they re to survive. Since the oxygen content of the host hemolymph does in fct decrese with the progress of prsitism, this would be n dvntgeous dpttion on the prt of the endoprsite, which, through its own development, progressively destroys its host. Since the totl dissolved oxygen content of norml host hemolymph t ny given time is of the order of 00 yl. nd the minimum mesured oxygen uptke per hour of young firstinstr Nemeritis lrv is bout equl to this figure (o'c^ooo /il./hr.) it is highly probble tht the prsitoid is utilizing ll the dissolved oxygen vilble to it. Once the prsite lrv hs estblished itself in the hemolymph of its host nd is respiring oxygen t rte in excess of the totl oxygen content of the hemolymph, its own respirtion becomes limited by the rte t which oxygen cn diffuse into the blood from the host's trchee. The progressive lowering of oxygen tension in the host hemolymph observed during the development of prsite (Fig. 2) suggests tht this rte of diffusion is the limiting fctor. The initil observtion tht the host cnnot support more thn one prsite unless the oxygen content of its hemolymph is rised rtificilly supports this explntion (Fisher, 96 b). The observtions tht superprsitism often results in prolongtion of the preimginl development of the survivor (Slt, 9; Simmonds, 9) nd tht suppressed lrve recover when trnsferred to fresh nd hitherto unprsitized hosts (Simmonds, 9; Fisher, 966) re lso in ccordnce with n interprettion of this kind. Clerly the vilbility of oxygen in the hemolymph of the host is importnt to the survivl of the prsite nd the evidence presented here strongly fvours sphyxition s the mechnism of physiologicl suppression. SUMMARY. The oxygen content of the hemolymph of mture lrve of Ephesti kuhmell hs been mesured in helthy individuls nd in those prsitized by the ichneumonid Nemeritis cnescens. In the ltter the oxygen content decreses with the progress of prsitism. 2. The respirtory rte of first, second nd thirdinstr lrve of Nemeritis hs been mesured nd found to increse rpidly t the end of the first instr, t bout the time t which the phenomenon of physiologicl suppression ppers.. The prtil pressure of oxygen in the gs mixture surrounding the Ephesti lrv ffects the oxygen content of its hemolymph proportionlly nd so ws used to lter the vilbility of oxygen to the prsite in vivo.. The survivl of Nemeritis lrve increses with the vilbility of oxygen in the hemolymph of its host. 5. The cpcity for survivl under conditions of low oxygen tension is miniml for eggs nd newly htched lrve of Nemeritis, but rpidly increses with ge. 6. The hypothesis of physiologicl suppression of supernumerry prsitoid lrve by sphyxition is discussed with respect to these observtions nd is held to fe supported by them.

5 RODERICK C. FISHER REFERENCES ADLER, G. (97). De l tension de l'oxygene dns le tiesus de quelques invert^bres. Sknd. Arch Physiol. 5, 662. BABERS, F. H. (98). An nlysis of the blood of the sixthinstr Southern Armyworm (Prodeni eridmd). J. Agric. Res. 57, 697706. FISHER, R. C. (96). A study in insect multiprsitism. I. Host selection nd oviposition. J. Exp. Biol. 8, 26776. FISHER, R. 0.(966). A study in insect multiprsitism. II. The mechnism nd control of competition for the host. J. Exp. Biol. 8, 60528. HOLTHR, H. (9). Technique of the Crtesin diver. C.R. Trv. Lb. CrLberg (5er. CUm.), 2, 0978. KROGH, A. (9). On the hydrosttic mechnism of the Corethr lrv, with n ccount of methods of microscopicl gs nlysis. Sknd. Arch. Phytiol. 25, 820. PROSSKR, C. L. (950) (ed.). Comprtive Animl Physiology, ist ed. Phildelphi: Sunders. PRYOR, M. G. M. (955). Inhibitors of Tyrosinse. J. Exp. Biol., 688. ROUGHTON, F. J. W. & SCHOLANDER, P. F. (9). Micro gsometric estimtion of the blood gses. J. Biol. Chem. 8, 550. ROUSCHAL, W. (90). Osmotische Werte wirbelloser Lndtiere und ihre dkologische Bedeutung. Z. toiss. zool. 5, 9628. SALT, G. (9). Experimentl studies in insect prsitism. II. Superprsitism. Proc. Roy. Soc. B, ", 5576. SIMMONDS, F. J. (9). Superprsitism in Nemeritis. Rev. Cnd. Biol., 58. SPENCER, H. (926). Biology of prsites nd hyperprsites of Aphids. Ann. Ent. Soc. Amer. 9, H95 THOMPSON, W. R. & PARKER, H. L. (90). Morphology nd biology of EuUmneri crsrifetnur, n importnt prsite of the cornborer. J. Agric. Res. 0, 25. TIMBERLAKB, P. H. (90). Observtions on the erly stges of two Aphidiine prsites of phids. Psyche, 7, 250. TIMBERLAJCB, P. H. (92). Experimentl prsitism; study in the biology of Limnerium vlidum Cresson. Bull. U.S. Bur. Ent. 9, pt 5, 792.