======~~=~:;~======~ TECHNICAL BULLETIN No. 230 ~ MARCH, 1931
UNITED STATES DEPARTMENT OF AGRICULTURE WASHINGTON, D. C.
MACROCENTRUS GIFUENSIS ASHMEAD, A POLYEMBRYONIC BRACONID PARA SITE IN THE EUROPEAN CORN BORER
By H. L. PARKER 1 Bntom%(Jist, Dirillioll of Ccrcal IIntI ]t'OI'a(JB n/"~cct8, BII/'eal£ of Entolll'()/o(Jy
CONTENTS
Pag~ Disrowr)' in Enrol'" lind id,·lttltr 01 th~ Biolog\'-Continued. ~I)el·i('.s _~ .. _",. _" ~ ~ ~ " ~ .. ,_," _.. ______.. ______... .. A v'emge ~ize 01 <'olon)' nnd proportion 01 Om1b'faphic distribution !lltd host fl·lations ... ;Sf'XCS... _~ ~ ~_~ ____ • __ • _____ ...... ____ .. _ ... __ 44 lIi;;tory 01 poIY~lllbr;'on)' in Hymenoptl'r!L .• Parthenogenesis_____ .. ______46 Iredu ic (ollowlld, __ , ... __ ~ ___ .. __ • ____ .. _* __ 0,. __ 6 Consideration of the zones in wi'ielt "\Iacro· ExpilLll!ltinn 01 symhols U~l'tl in thl' illu,tr!l' ceni"'3 gifuen.i3 o~curs ...... _...... 47 tlOns...... __ ...• 7 l>hysil'l\l chllructcristics ___ ... _...... _. __ .... ""'__ _ 47 Descriptinn 01 .'[(lcroctlll",. ui.{ue/l~is .._"'" 8 CliInct·c l'h9ractcri;;tics ______.. ______.. 4~ The !ldult...... 8 Seasonlll histor)'.... _...... _...... 49 'l'he l~gg. __ • ___ ~ __ ~~_ .. ____ ~ __ ~ .. __ . _.~_ .. _ 9 ""Ilsonnl h!story 01 the host...... 49 1"rhl'lnrvLL. __ .... ___ ...... ___._.o ___ ...... 9 Sellsonal hIstory of the pI r!lSite...... 49 rl'hc pnlJ)upn unu pUpil...... ~ __ .. ~ .. " ... __ _ 23 ESll('rimcntal dutB.. ______.... __ _ 51 'PI10 cocoon_____ .._.... _.... _.. __ ~. __ .... ~" .. * _. __ 23 Laboratury breeding l'xIlcr'luents____ ,.. __ _ 52 .Bit)lo~y...... __ ._...... 24 J\Iort"lity in renring 01 field materiaL_.. 52 EtIu.\r~l·nL't·. __ .. ~ .... _ ...... __ ... __ ~_~_~ ... _ 24 Summary of duration 01 \'DriollS stages.. 53 L,ght rmctions .•... __ ...... ~4 Hapidi!y 01 spring dcn·lopment...... 53 Feeding...... __ ...... __ ..... 24 Mortality ill the prcpupal pnd pupal Oogenesis____ M _. ~_. _ .. _ .. , _". ~ _~ •• ~. 4 .... _ .. 24 stages..."...... 55 2-l Cnld'storage 01 o\'crwintp:ing host larvae ~~\~~~~~\W~':i:.~:: ~:::::::::: :::::::::::::: 25 contl\illing parasites ...... _...... _ 55 ~t:gml'ntation of tho (I~g _. " ...... ~ ~ 29 Cold slorage 01 cocoons...... 56 Growth and dcv\·lopull'rt.. of tlw l'~g ~_. 0" :10 .\Iacrocentru. gifuen3i. as !I co: DISCOVERY IN EUROPE AND IDENTITY OF THE SPECIES 11laCI'OCentJ'tt8 gilllen~i8 Ashm.,~ a primary parasite in the lal'va of the European corn borer (Py1'a'l1.~ta. mtbiZalis Hiibn.), was found in common mugwort. A1'temi8ia 1)ul gads L. (99),3 in the Armorican zone of w('stern France by 'Yo Gamkrelidze. of this laboratory. while on an exploration trip during the summer of 1925. . 1 ThE~ work reported in this hllIletln wns done at tb~ Europcuu Pnrnslte Lnboratory of the llurenu of Entomology at Hyl)[·cs. Val'. France. from April, 1:)27, to October. 1929. :I~he writer expresses his grntitlld~ to Pnui '\Inrcilltl for examining und criticizing the microscopic prl'purnti(llts and llrawlngs; to C. F. W. '\hU'sebeck and A. II. Guhun for help with the identity nnd de~criptioll of this SPl'l'les; to Esther Hart for her drawings of the adult nnd cocoons; to W. U. Wnlton. ,Yo H. Thompson, nnd E. UnbnIH] for ht'lp wltll the mnnuscrlpt.; tn Y. '\lnlamn for the photomlcrogrnph;;; nnll to A. 111. Vance, II. D. S,,;lth. nlll] '1'h. lI[nlnmll fllr h1'11> In \'nl'lons wnys. • Famlly Hrnconlclne, order !. ~·menoptera. class InBI'ctn. 3 Italic nlllllUCl'S in pnl'l.·nthl"CS ref(,l' to Litl~mtlll'e l'ltl~d. p. 01. 1 :!40S1-:lI--l 2 TECHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE Further study by the writer soon revealed the fact that this species was an important primary parasite, and exporta.tions to the United States for colonization were begun the following year. At first it was believed that this species was .ill. abdmninalis Fab., and in an earlier publication (129) it was so designated. The writer has not examined the type of M. abdOininalis, but. A. B. Gahan has compared speeimens from P. nuUlalis with the type of M. qifuensis and believes that they are the same species, while he considers M. abdonl:inalis to be a distinct species. GEOGRAPHIC DISTRIBUTION AND HOST RELATIONS This species has been reared by the writer from host larvae taken from Artemisia in the Sequanian (south), Armorican, and Central zones in France, and from corn in the Danaprisian (north) zone in Russin. 'V. B. Cartwright has reared it in Japan from P. nubilalis taken in col'll and hemp. Hergula (7) has bred from larvae taken from COl'll nt Kraljevec and Bjelovar in Jugoslavia a species of Muerocentl'us which he claims is identical with that bred from Tours material. It is qnit(' likely thnt this is also M. gifuens'is. So far a<~ .is known it has not been reared from other hosts than P. n'llbilalu. It is possible, however, that M. g-ifuensis has often been misidentified as ill. abdomi1uilis and that, consequently, some of the biological and descriptive data in publications on M. abdom 'inalis pertain in reality to M. gifuensis. As there is no definite proof of this condition at present, such data can not be definitely incorpo rated in a paper devoted exclusively to M. gifuensis. HISTORY OF POLYEMBRYONY IN HYMENOPTERA The discovery of polyemhryonic development in the parasitic Hymenoptera by Paul Marchal and his subsequent studies and those of F. Silvestri are so well known and have been cited so often that it is not necessary to go into detail concerning these matters. For the purpose of completeness. however, it is considered desir able to cite here the forms known to reproduce by polyembryony as well as those suspected of such reproduction. It is pointed out that most workers on the Hymenoptera suspect that a great many of the vast group of Enl!yrtidae, whose biology is as yet unknown, will be found to reproduce in this manner. Polyembryony in the Braconidae,4 more especially in the genus Macrocentrus, was suspected as early as 1913 by Cushman (3). In 1927 Vonkassovitch (30) supposed 1t to exist in ill. abdorninalis, a parasite of Psal1wwtis ltyalinalis Hbn. Later, Thompson and Parker (:89) held it probable that M. gifuellsls (then erroneously called ill. abdo111:i.nal'is) reproduced in this manner. This bulletin leaves llnt'!arifiec1 many aspects of the reproductive activities of ill. gifuen,w,s,' such as oogenesis and the origin of the germ-cell determinant. the maturation divisions of the female pro • Fink (5) describt's the transformation and change in shape of the el-;g of AI. ancy/i-vora, hnt hl' gh·es no datil on the segmentation (II' the structure (If th., g .. rm~ ,md embryonic m'~mbrull"s. After Il ('nreful study of Jj'ink's ren:urks (In the sup..rparusitib.... !'tc.• In this species the writer considers It possible thnt tlJf~rl' Is poly"mhryollY in..ll' '"LCIflivo/'U. 1n nil ('Yents th!' mutt"r should hI.' iIlYl'stigut("I more closely. l\IACROCENTRUS GIFUENSIS, A POLYEMBRYONIC PARASITE 3 nucleus, the CC!ljugation of the male and female pronuclei, the COIl jugation of the germ-cell determinant. and early divisions of the primury cleavage nucleus. 5 The writer holds. however, that he has demonstrated the following points: (1) The differentiation of the egg into a pregerm composed of a paranudeus (pl'oLably derived from the polar nuclei) which directs and governs the trophamnios, and germinative or true embry onic cells which give rise to embryos; (2) the division by fission of the primary germ accompanied by a division of the paranucleus to form the secondary germs, which again divide to form morulae, which demonstrate (3) polyembryony in M. gifuerusis . .A. summa.ry of our knowledge of polyembryony among the para sitic Hymenoptera is presented in Tables 1 and 2. • 'l'hi~ phn~e (If denJoplIll'llt will be mude the subjpct of further researches. ~ TABLE i.-Parasitic Hymenoptera /;nown to reproduce by polyembryony --~r--· ;;; Xumber t':I ! formed o £rOIllone III Authority for polyem· 'I' nrief summary of details and peculiarities of development Pumsite spe~ies lIos! egg or sl~t~!~I,~~;,Yryi ~ hryony in the spedes , issuing ..... ,from one n [ host :. I C" ,------ t:;j ('halcidoide:1.' .\gcniospis fuscicollis :\lllrehnl (I.;) Hyponomeutus mnli- ! 150 Polar bodies form paranuclei which (in trophamnios) encircle germ cells at or q (Dulrn.) 'l'h. nellus et al. shortly ufter first clcuvage. BIastomeres become grouped in many distinct groups forming morulae. Morull\3 divide b~'fission. Proliferation of bost ~ tissue to form a nucleated membmne whIch incloses all morulae and elongate:; t':I in form of B chain, No dissociation of parasite bodr. Develops in "iscernl ;;; ea\'ily. Do••. _ .\, fuscicollis praysin· Silvestri (25). __ ., Prays oteellus Fnll.• 10 Similar, except that paranuclei from polar bodies do not encircle germinati\'e cells ~ coin Sih'estri. ' until 12-celled stage. Do. , I.itomllstix trunlO\tel· :;ih'estri (E.I) •• _ PlusingumrIl!l L l,liO() :legmenting egg differentiates into two distinct parts; one part from the polar c-:'" Ius Dnlm. bodies forms a nucleated membrane, the trophamnios, and incloses tbo other o part, the blastomeres. Tbese become grouped in many groups of about 6 cells, forming polymorulae. Tbe whole mass l~broken up into many polyruorulne and isolated morulae and tbese are distributed throughout the host's body. ~ Hacb morula or polymorula remains Inclosed by a part of the nucleated meIll· home (;, e., trophnrunios with its go\'eming pamnuc1ei). In addition to sexual ~ larvae, a.'exual or monster larvae are formed which do not develop to maturity. De\'elop, in visceral cavity. Cl t':I LithocolletL~cramer. Ii 12-15 Advanced stages found in cyst ofhost tissue similar to that caused by A. fusclcollis. 1)0 ••••.• Ageniaspis testoceipes :\Iorcllul (15) ...... I-d ]illtzb. I elln, Do_•.••• ('opidosoma bnyssoni ' Sih'estri (£6, Coleophoru stefonii j 41-11~Probably similar to A. fuscicollis. I'aranuclei do not encircle germinative cells !"l :\Inyr. Jonn. , before 14-celled stage. o 1)0 ... ('opidosoma nanellne : Sih'ssUi (E8.). ' Recnn'arin nan ella I 3-11 Embryonic chains and asexuallar\'ac observed by Silvestri. >tj :-iii\'. I IIilbn. I 1)0. _ .. _., ('opidosorna geleehill. Leiby (10), Patterson IG norinlOschemll gal 165 Polar bodies form paranuclei. Blastomeres become grouped in groups of about :: lIo\\,. (:?O). Illmolidoginis Hil. I 6 cells; troplLqmnios with its paranuclei incloses whole body. Parasite body o dissociates into isolated morulae or polymorulae of various sizes, each remaining :=: inclosed by part of trophamnl05. Morulae divicle by fission. Develops in ...... visceral cavity. n ])0 •• _._. l'opidosuma bonchtL Parker (19)"_' __ __1 Oelechin pingninelln 38 Develops in \'isceral cavity in chains resembling those of A. fusricollis. Asemnl "" ~ anUIU Rlltz. '!'reits. larvae pr~ent. ;;; ])0 ••••• _ Copidosoma thomp· ,.••.. do..••.• _.•.•••.•. _ Nothrix senticetella j 18 Similar to C. boucheanum. ::\[",Ie unknown. q soni 1\[erc. I Stgr. :::l DIl_ ••••• I.itomnstixkriechbnn.: Ferriere W ...... " ... Depressaria alPigen'l 21-121) Chains of developing embry~sobserved in hody ca\'ity. merIMllyr.' ella Frey. t':I 10-12 morulustage the peripheral cells become detached to form the paranuclei of tbe Serphoidea•. I'olygnotus minutu~:\Iarcbal (15) .... _.... Phytophagu destruc· , In Limlrn. tor Say. tropbamnios. The remaining cells in the center become grouped in 5 or 6 main groUP3, all remaining inclosed by the trophamnios. Each group so formed give:; I rise to a larva. Develops in gastric sac. P"" ~- Do___•.. ' Pilltygaster vernalis; Leibyano Hill (OJ•. _ :._ ••• 00••. ____•.• _•.•.• '- 8 ; Two I)ol"r bodies formed, which constitute the original parunudenr masses or Myers. I i governing nuclei of tbe tropbamnios. The cleavage nucleus dh'ides four times, tbus giving rise to 16 cells, each of wbich apparently produces an embryo-forming I I germ. The germ, remain together surronnded by the trollhamnios. Some of them may di\'ide once by tIs.,ion. Development takes place in gllStric sac. !;J ..... Vo._._ 1'Iatyg'l~terhiemali~ ! Leib~'ano Hill (I1) ••• I.....do.•__.•..•...••.•. I or 2 ; 1'11'0 polar bodies are formed whicb nnite to form the paranucleus of the tropham· Forbes. nios. Clea"age nucleus gives rise to a 4-celled germ wbich divides in 2 daughter > ! I germs, each germ taking 2 cleavage cells. Paranucleus ui\'ides to suppl~'each n , I daughter germ. The 2 daughter germs separate and give rise to 2 embryos. :::l De\'elopment takes plat'e in visceral cavity. 'l'bis species may also develop by o I, n monembryony and is considered an example of the most primili\-e method of t:j polyem bryonic development. 15 Z \'ariahilis Leihy (I1)- ••-- •... ' •. ~iHhopalomyill mrolin;l. Early stages not elucidated. DO"-"' i PI~~J:ter H Braconidae.", l\Iacrocentrus gifuen· Parker (present bul, , pyrausta nubilalis 10 ' Early stages of cggsegmentation and formation of pregerms seem to resemble those :::: sis _\shm. Hbn. of A. fuscicollis; primary germ di\'ides by fission to form 2 secondary germs. -::: letin). I Paranucleus divides and balf accompanies each germ. Tbe paranuclear cells rr. multiply by amitosis and yia the tropbamnios they e\'entually encircle com· o pletely the germinative center. Tbe secondary germs again divide to form tertiary germs or morulae, wbleb by multiplication of embryonic cells transform ~ to embryos, General dissociation (,f germs; some remain grouped in bodies of q 2 l :;: :Dryinidae __• Aphelopns theliae; Kornhauser (9)._. __•. _\ T~~\\'abimaculala . __ ••.•.•• 1, Gahan.; Fab. rr. Bracondsc__•. l\Iacrocentrus abdom· 'I Voukassovitch (,'1)',. __ Psammotis hyalinalis 24 to 29•• Observations on size of colonies, sexes of adults in colonies, etc., indicate that ~ inalis Fab. I Bbn. et 81. I polyembryony is probable. t;"l CJ1 6 TECHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE TECHNIC FOLLOWED The parasitized hpst la.rvae were collected and kept for storage und rearing purposes in corrugated paper in copper cans, a system devised by Jones (8) for shipping and rearing. Cont.act mOlsture was supplied when necessary by plunging the cnns in water. The colonies of Macrocentrus cocoons spun in these cans were removed to glass vials for observations and supply. Adults were kept in several sizes of lamp globes seated on a: cloth base and in glass vials. Moist cotton in a glass dish was placed on the floor of each cage and wet cotton wads were u.ttached to the undel'side of the screell-wire top. Food wus supplied by smearing gl'llllulated honey on a small area on the wall of each cage, over which distilled wuter wus dropped ut intervals; distilled wuter for drinking was spmyed on the wulls. Dry incubutors at 18°, 20°, and 25° C. were used in all experi ments and an electric refrigerator at 4° to 8° C. ,yas employed in the cold experiments. For oviposition purposes it was found necessary to confine the young lllrvl1e for the reason that a larva free to move usually escaped from nn attacking female without receiving a thrust. This condition, combined with the small size of the egg and the ease with which it could be lodged or concealed in the host organs, required t.lUlt a lurvll be stung several times before Ull egg could be recovered, as lurvae stung only once 01' twice seldom 01' never yielded an egg by ~imple ~lissection in physiological media. This confinement was Ilccompltshed by "sewmg" the larva down to the cloth base of the cage with u. fine wire or thread. A piece of leaf with several small holes in it was placed over the larva. A smull amount of la,rval frass was then smeared oyer the npper surface of the leaf to excite the females to oviposition. In this way, as the oyipositot· was thrust into the holes it would generally encounter the fixed host larva aml oviposition would result, A.fter 10 to 15 thrusts, dissection of the larVlt woulclllsually yil'lcllln e:,rg ot' sometimes two or three. Laryae so impaled wer~ usdess lOt' rearing purposes, however. This technic WilS later improv{ld in tllt' following manner: Small larvae were isolated in gIas:-; tllbe~' togf'ther withn tiny stem or cube of It food plllIlt, such as mugwOI't, dock, or com. After the larva had burrowed into it for about the If:'ngth of its body, this stem with the contained larva, was placed in the cn.:,re to receive oviposition. In this way it wal; possiule to procUI'C n considcra.ble numuer of thrusts in a single larva. The oniri(\S were studied by c1issection and simple obsermtion ;md hy se~tioning ill Plll'llffin both within the abdomen and removed from It. Eggs were studied by direct microscopic observation with and without stain!': :l.ud by seetioning parasitized host hUTae. Parasite bodies anll germs in all (recorded) stages were studied by direet obsernltions. stainer} und unstained. and by sectioning both within the hO~5t and free from it. 'rhe Hxing fluids mostly <.'ll1ployed were Bonin·s. Duboscq-Brasil. Cal'T:ots~ Ca.rnoy and I.e Brun's. :!nc1 Flemmin~'s without ncetic acicf BOIHIl H p1'o\'e<1 to be the most satIsfactory by fat'. Staillls uRed were alcohol-earmin and carmalum for entire bodies. J!lydH'malulIl and eo;.;ine. trichromique de Cajal. iron hematoxylin: ~ l\:t:AOHOCENTRUS GIFUENSIS, A POLYEMBHYONIC PARASITE 7 orange G, und Congo red and hemnlull1. The most satisfactory method wns to use acid glychemalum followed by eosine. :Fol' toPogrnl)hieal studies of the internal organs, muscles, etc., lal'vne were fixed in Carnoy's und kept on H5 per cent alcohol. The desil'ed orgnlls were then dissected out with fine needles. The histo logicnl obsel'vlltions and illustrations were made from specimens fixed in Bonin's and then sectioned, and stained in glycheuutlum and eosine, It is here pointed out that the tissues of parasitic larvl1e are mor FIGt:UE 1.-.\dult f~mnlc of Muc/,oce"tI'l18 yf/U(1I8i8. X 10 above brown or bluckish, sometimes paler at the suture;:, Yuriable, the first, second, aud third se/,,'1llents lonlr1t.udinully striated; antennac very long, brown ish yellow, becoming dusky toward apex, 40 or more jointed, the joints dusky MACROCENTRI:S GIFUENSIS, A POLYEl\IBRYONIC PARASITE 9 at apex, npPl'Hl'lng annuluteli; legs, including coxae, yellow. \Vlngs hyaline, the veins brownish, the ::;tigmlL with a large dal:k brown blotch \\-ithill. 7'/I/iC.-Cllt. No. 72H, U.S.N.l\1. Lo()(/Ufl/.-Gifu (Y. Nawa). ~'wo spedmens. M. {riflu:nsis Is l'a:::ilr (listinguish('ll from the other purasites of the corn borer by its sleU(let~, delLeate (orm, it::; light-brown color, long antennae, and the HlenlieL' ovIpositor of the {('mille. OVARIES The ovaries lll'e of the meroistic type and are bunched in two gl'OUPS, one on each side. Then' are usually 1~ to 15 ovarian tubes in ('a('h group, though in some individuals there arc as few as 8. The t.ubes of l'tleh group nre joilled togethel' at awir bases and empty into Hhod oyiducts; the!:ie unite to form a shod ,-agina to which the black ish spermlltheca. and the various glands common to the Braeonidae (6, 18) nrc Ilttnchel1. In some cases the two oviducts are joined. superficially at lClH;t, at the base of the onn'ian tubes, separating immediately below this point and ('olllin/! together again into the vagina, thus forming It sort of loop. It is improbable. }wwe\-er, that thcl'l! is a Illutuallllmen at this distal jllnetion. THE EGG The OVllrian and newly laid ('gg (fig. 2, A. B) lIleasures from 0.08 to 0:1:2 mill. long by 0.028 to O.05G IIIIll. ,ride; it \'l1ril'S sonwwhat .in shape but in general is more or less ovoid. with one end (the end directed dowllwal'(ls in the o\'11 ry) FIO{JIlE :!.-Eggs of ,![ac/'OCC/I trlt.~ g;fll' slightly pointed. It has a large lludeus near tll1~ CII8;8: A, Ovarilln or newly Inlll l'gg: l't'nter awl a nudeuslike mass of dense granular B, another fornl of the ovarlnn l'gg. lIlah'rial at the lower end (fig. 2, A). which is The nuclei are in probably the. genn-C'pll SI7.El, COLOR, .\,,1> }'OR~( I..ellf!th of 11 ":ly hatched lar\'l1, 1.5 mm.; width, 0.:28 111m.; width of head, 0.298 1I1In.; length of mandibles, 0.01)54 ll1m.; length of larva after feeding, 3 llIlll.; width, 0.43 mm. (Fig. 3, A, B.) The colol' is translucent grayish white, the head ('oncolorous with the body. Tho larva of this stage is composed of a large, somewhat flattened hl'ad with llrotrllding. distinctly delineated buccal regions and 13 well-defined bOlly segments bearing four rows of prominent lobes resembling pseuc1opo(h~. EXTEUX AL ~\.XA,[O~IY The head is somewhat more heavily chitinized than the body seg nwnts, and antel'iorly the Ill'shy mouth parts protrude as if "over "Fink (5) IUl6 til'scrll>ctl the tlrst·stn~" Inrvll or M. a/lel/lieora. His 1'igllre -l r('sembkR but little his (lhoto~ruphs (tl~. :~. U) of the flrst·stn~e Inn·n. It would not be BurprlslnJ; to flnll thnt the newly hatchcd hll'\'a of .If. allcJllicora Is very similar to thnt of .ll. !JiJIIVIIHi8. !:!.JOS1-:n--~ 10 TECHNICAL BULLE~'IN 230, U; S. DEPT. OF AGRICULTURE flowing" fl'om the more solid skeleton of the head. These parts include 11 distinct labral region having two sepl1rate sensory papillae (fig. 3, 0, sp) and two very prominent conical protrusions or labral proceS~ies (fig. 3, 0, D, lbrp), each of which has five sensorial pa pillae; the labium (fig. :3, C, lb), which has two similar protrusions \I JI lo'l ...,' Fde~ FIIlUIlH 4.-Mundlhh~H und truclH'nl sy~t,'m of IlIr\'lll' of MIWroCCllt,.//8 gitUP-IISiN: .\. Mantlihl{' of flrHt-Htllgt' lur\,a'\ B, mUll(lIhle or St~(~ond-stuge larva; C. muntllllie of third-stnge Inr\'n: D, tmchen sYHtem of flrst-Htnge Inr\'a; E, segment tlC body of flrst-Rtnge 1111'\,11 ttl show origin lint! dlrecthHl of IlrineiplIl tmellenl brunellI's; " nud ,. nrt' hl'lInch,'H t'xtl'ntling inwllrtlly. lind Clint! tl IIrt' brnnches extelltling outwllrtllr: F, trlldlelll brunehl's fl'uin middle of nnterior !'ommisHurl! of first-stnge IlIr\,1l Hegnl('lIt XIII. whieh is curved call dad. The dorsolatel'Ul pail' on H.pgment XIII is inconspicllom; or absent in some specimens; Seg ment, XIII is conieully pointed posteriorly and bean; dorsally the evaginated rectum. Thel'e are no tegumentHry spines 01' setae. I ""fER:,,(Ar. Al"A'fOMY The digestiYl' tl'Uet commences at the mouth; the esophageal pump 01' pharynx is loeutetl immediately posterior to the mouth and the 12 TBCRNlCAL BULLETIX ~30, U. S. DEPT. OF AGRICULTURE esophagus connects this organ with the mid-intestine into which it empties in Segment II. At this point there ill an esophageal valve WhICh prevents the regurgitation of the food. (Fig. 5, A, esv.) The -. -_..... lhr p .'..-... 1nd o FlGUIlE G.-Second, third, lind fourth stages nnd head parts of lan'ae of MaC,.ocellt""~ gi(1I01l8i8: A.-Second·stnge l:um In outline, sliowing digestive tract. The ~all\"llry glands and ~Ialplghinn tubes arc IIbbrevlated. B.-Thlrd·stage larvlI, side view. C.-!.'ourth·stage larva. D.-Cross section through hend of second· stage larva. E.-Verticlll section through head of lnrva just in front of brain mid-intestine before feeding is a slender cylindrical tube; a.iter feed.. jng it is distended and slightly more oyal in shape and is filled with a. creamy yellowish, granular substance consisting of fat globules in the process of being digested. Xear the anterior end tlie short :ff.tAC110CENTltUS GIFUENSIS, A rOLYE:i\rmWONIO PARASITE 13 peritl'ophic membrane (pm,) in eurly stages of development can be seen inclosing the UlHlig-ested food. This membrane is probably secreted by the group of special cells (so) situated in the anterior end of the mId-intestine und grows slowly until it fills most of the stomach. In!t gl'OUp of young InrY1\e the pel'itrophic membrane in all stal'es of deyelopment may be easily observed and dissected out. No undigested food is e\'e1' observed outside of it in nny larvnl stage. In segment XII the mid-intestine joinH but doeH not communicate with the hind-intestine. This orgnn iH in two distinct parts, the ileo ('olon to which the :Mnlpi~hian tubes are attnched and the rectum which is eyaginated in tlllS stage.' A pair of simple l\fnlpighian tllbes arise from the nnterior portion of the ileo-colon and extend lorwllrd vent.rnlly, the right one to Segment IX, the left one to Seg ment X, though sometimes the lett one is the longel'. A p!tir of ramified sn.livltl'Y glands empty into It very short common duct in the head.s Fl'Om this point posteriorly, ench gland rnns along the ven trul floor for It shut't distance, rises somewhat, and again brunches into two prineipnl trunks in Seg-ment IV. Each of these trunks sub sequently mmifies in all irregulllr manner into dorsal branches of nlrying length. The tl'llchenl system (fig. 4, D) is composed of two latera,llongi tutlinul trunks cOllnected anterior! V by 11 dorsal transverse commis snre located in the first segment 1I1Hl 'posteriorly by It ventral com missure ill Segment XIII. Itnmifying branches extend to the he::td from both the anterior commissllre and the lateral trunks. In ench :,;egment threc lateral branches arise from each of the main trunks; the first. two of these supply principally the inner orgnns, one extend ing dorsally and the other ventmll~r, while the third branch supplies the outel' organs, such ns muscles, fat, nnd hypodermis. 'fhis branch (fig. 4, E, c, d) Tuns in n, ventrolateml direction for It short distance and separates into two parts, one part supplying the yentrolaternl lobe and the ot.her supplying the dorsolateral lobe. Each of the.se subbranr.hes is, howevCl', subsequently ramified many times, and these l'mn.ller branches wander llbout from the dorsal lobe to the ventral one IWlI back again. The lobes as well ns the body proper have many tl'llchelll terminations in the hypodermallnyer, but there are no morc trncheae or tracheal terminatiolls in these lobes than there Itre in the other parts of the body surface, i. e.. in the body cavity between these lobes Imd in the henet. Segment XIII has fewer tracheae than the other seg-ments; the e"aginnted rectnm has no trncheae, Ther'~ are no spiracles. - The nervous system consists of a 2·lobecl brain, t.he subesophageal ganglion, and 12 distinct segmentnl ganglin,; la.ternl bmnches arise in each ventral ganglion. The fat body is not conspicuous. though the small groups of fat cells mn,y he seen between the musclIlar lav(ol' and the digestive tract. The reproductive organs nre representee1 by two small ()'ml gonads locuted in the posterior part of the vis('('1'Hl cavity, -----.-~.------"J'h.ls Ilrutl't1~IOII Te!!umentary~ ~ muscles ca.n be observed extendinO"• 0 from se(~ment0 to seg.ment, but ,none are present in the lateral lobes. They are de st'nbed mOl'e fully for the fourth sta~e. The circulatory system is of the u~ual hymenopterous type. (See de~cripti.on of the fourth-stage larva.) The lustoblasts of the adult external appendages, such as win as, legs, and reproductive organs. eun be distinguished as thickellirwsin the hypodermis. ' . 0 The fully fed larva differs little from the newly hatched larva (·xcept that it is larger und slightly swollen, the stomach content:; "how prominently, and the lateral lobes are less conspicuous. During the molting process the old head capsule SI)lits, the head is li~ted out, ~nd the cust skin sl.ips backwards ventral y, ?ften being )JlII'tLally retamed, howe\-er, untIl the laryu reaches the thu'd or even fOil rth stage. SECOND STAGE Lenhrth or newly-molted larva 2.08 IIlIll.. width 0.4 llll1l. (length afh>r feeding 3.8 mm.). width of head 0.3 mm. (Fig. 5, A.) The color is the same as in the first stage, but less transparent. The second-stage larva is composed of a somewhat globular head amI 13 body segments; the general form is cylindrical, tapering l"lightly anteriorly and postel'lorly; the last segment is pointed and belll's dorsally the evaginated rectum.9 Slight ventrolateral and dorsolateral bulges are present where the lobes existed in the previous iitage. There are no body spines or sensorial papillae or spiracles. The head is scarcely more heavily chitinized than the body, The lllouth parts are distinctly delineated. There are no mandibles; the points where they will later appear are represented by fleshlike lobes. (Fig. 5, D, mel.) Sensory papillae are present on the various mouth areas. The internal anatomy is much the same as in the first 5iage, with the exception of the following points: The mid-intestine has increased in size and fills the greater part of the body cavity. The peritrophic membrane with its contents fills about half of the mid-intestine; the fat body is developing by the increase of the size of its cells, inter spersed among which nre numbers of urate cells with their granular white contents showing prominently. THIRD STAGE Length 3.5 mm., width 0.6 mm., length of chitinized tip of man dible 0.56 mm. (Fig. 5. B.) The larva of this stage, while somewhat similar to that of the second stage, is proportionally less elongate and resembles more in general conformation the typical hymenopteriform larva. Segment XIII is shorter and the rectum is not or but little evaginated. The mandibles {flu. 4, B) have reappeared and are simple and rather blunt, very ligiltly chitinized, and have a rounded, slightly projecting il're~ularity on the posterior edge which might be called a tooth. The°irreaularity is situated at a point coincident to the most distal of the re~l teetli which appear on the fourth-stage mandil.lle. There al'e no spiracles; the body is without spines or sensorial setne. • Fink (5), in his illustrntion of the second-stage larvn. shows the annI opening at thc 4'xb'emc end of the tenth abdolcinnl segment, and only part of the rectum evaginated. In .If. (lifllenBis the 1I11al opening is dorsad of the termlnnl pOint of tile tenth segment and the 'whole of the rectum-up to the point where It joins the i1eo·colon-is cvaglnllted. 1\IACnOCENTUUS GIFUENSIS, A POLYEMBRYONIC PARASITE 15 FOURTH STAGE SIZE, COLOR, AND l!~ORM Length 4.5 mill., wiflth 1 to £ mm., width of head 0.5 mm., length of mandible 0.0937 ll1m., width of thoracic spiracle 0.0375 mID., Ilbd(lminlll spirueles 0.0281mll1. (Fig. 5, C.) lnplp (,"11:1.:111: H,-Cells, lliUHcll'S, IIl1d IWlld pnrts of hu'vlIc lit .UUGI'Ot'/'lItl'/lH lIifuel/x/x: A. Cl'Ils IIf \'lIrlOUS t)rgllnH of till' III 1'\'11, lIll drllWII to thl.' Huml' Heulc (Boulll'H, gly ~hemlliulli); B, musculnturc or Inn'lI, Segments I, II, III, IV, XII, 111111 III; C, Hegml'lIt of I!pllrt, showing Dlusculllr tlbl'I'" lind pl'ricurlllni C..U8; D, front vil'w of 111'1111 of fourth-stu"I' lurnl : E, ~Ide \'Iew or Iwnd with hulf (Illcluding the snlh-llry "llInlls) cut II\\,II~' The color i!; a trall:,hll'l'nt grayish white, with creal1ly-yellowish mid-intestine, fut bodY, lind white urate cells pl'ominent. The lurva. of thi~ 'stage is of the typical hymenopterous shape, being ('omposl:'(\ of u ht'!Hl and 1;3 body segments. It is arched dor sally find is widest Ill'HI' the middle. The lal'ge lateral 10bN; due to transverse. tegunH-'lltal'Y muscles (fig, 6, B. t 2-5) in each abdominal sl'gment gl\'(' it :I HIIIH'what Hattl'lIed Hppearan('e. Itl T.ECHNICAL Bl.'LLETIN 230, U. S. DEPT. OF AGRICULTURE The prin<:ipal diil'erence in the general ,aspect of the laxva of this stage as compared with the larva. of the third stage is that the lateral lobes are mOrc pronounced, the ura.te cells SJ:C larger, !lnd the ~, an tennae," mandibles, spimcles, and tegumentary spines are visible under low-power fnll.gnificution, HEAD The head is roughly ht!lll't shaped in outline with two pllirs of short tubercular protuberances, or antennal rudiments, located in large circular elevation!:> dorsoluterally and somewhat anteriorly. Tbe bllccall'egions are clearly defined and consist of n, labrum (fig.. G, D, 101'), two prominent mllxillae (11101:) in 'which the mala is quite distinct from the cardo-stipeal region (1l.1"ho!lgh this detail is not dearly shown jn the drawing), and the 1abium (lb), which is e.ven more distinctly delint'A'lted than in previous stages and bears four pairs of sensory papillae. At the apex of the labium is the opening of the salivary duct containing the spinneret (s:;). On the mentum, 01' area below the maxillary and labial regions, there are two pa.irs of slllall sensory spines. The mandibles (fig. 4, C) are larger, sharper, and more heavily ("hitinized than in the preyions stage and they are provided with 17 to 20 small te(~th on the posterior (cutting) edge, The tentorium ext,ends across the. lower part of the head near the posterior margin llni,i holds the walls rigidly together. The mandibles articulate upon two llairs of ramal' (inferior and superior) of the epistoma. SKELETON The skeleton is composed of a rather thin layer of compact cuticle, It is flexible and tough and folds back and forth upon itself at the intersegmental lines and other points of muscular attachments. The ("utide of the head and tentorium if· thicker and more rigid than that of the body. CeTICL'I~AR SPINES SegnH'ut I bears 11 wide.band of small cuticular spines completely encircling it, with a small median dorsal patch extending posteriorly to join the band on Segment II; the individual spines in this area as well' as those on the margin of the armed band are smaller than the !>pines near the middle of the band; the spines near the anterior border of the segment are inclined somewhat posteriorly and those on the posterior margin are inclined anteriorly; the intersegmental lines are l>l1I'(,. S('gment II has a like band encircling the dorsal and lateral l3Uria ces, while immediately around the spiracles is a small unarmed areu. Segment III has II similar band but it is narrower and does not extend so far ventral'ly as in Segment II; though there are no spira ell.'S in this segment, the "Bpiracular" area is without spines as in Segment II. Segment IY is similar, but the armed band does not extend so far ,"entmlly 11lld the dorsal spined at'ea connecting the band of Segm('nt IV with that of Segment Y is smaller and has fewer spines. In Segment \T the armed band is diminiBhed in width dor- ~ sally and is only a nat'l'OW strip extending along the anterior margin of the segment, and the dorsolateral areas only are cO'"ered by the l\IACROCENTRUS GIFUENSIS, A POLYE.MBRYONIC PARASITE 17 spines. In Se~ll1ent VI only the dorsolateral areas are armed, Seg ments VII to XI are similar, whil'e in Segment XII these areas are reunited dorsally to form the band covering the dorsal and lateral areas. Segmpnt XIII is bare. S~:SSORIAL SETAEl Each segment except XIII has a number of ~mall sensorial setae inh'l"spersed umong the tegumentury spines. these setae varying jn number from about 40 on Segment I to 32 on the abdominal segment'>. They ure not constant in number or locution on any two segments of a single individuu.l nor on analogous segments of two different speci mens. The only constant arrangement obsened is that on Segment I there is a more or less irregular row of these setae ventrally on the l'eft side near the anterior margin of the spined band, while on the right side the l'olTcsponding ~roup is neat· the middle of the armed bund. Isn:ItXAL ANATOlLY HYl'OIlERMIS The hypollermis is a thin secreting layer just underneath the cu tide. It is ullifonn in thickness save ill the head, where it is con siderably thickened, especially around the mouth parts and the top of the head and lit several points where the histoblasts will appear. The cells of the hypodermis (Hg. (j 'A, 11.11) are regular in size, m?re 01' less hexagonal in shape, but globUlar when spnrsely placed, WIth ~1istinct ami easily stained round nuciei. In the thicker parts of this layer the cells ha.\'e their nuclei in different planes, thus giving the appearance of se\'eral strata of cells. The cells of the esophagus and of the posterior part of the rectum, as ·well as the imaginal l\Ialpighian tube buds and those of the extreme anterior end of the salivary glands, are similar to the hypodermal cells. HISTOBLAST!; The histoulasts, or imaginal disks as they are often called (in ref erence to adult appendages), are those cells of the hypodermis which become Liilferentiated uy their adivities more or less early in the larmllife to produce certain organs of the adult. At first these histoblasts are represented by a slight thickening of the hypodermal layer, but ·when the larva has reached full growth the cells have multiplied and pulled away from the cuticle, leaving it bare at that point, and the organs begin to form. The most con spicuous ones are the antennal histoblasts, which are situated in the head, one on each side and slightly behind the labrum (fig. 5, E, im..da); the leg disks, which are located ventrally in Segments I, II, anel III; the winO' pads, which are located laterally on Segments II und III; and the histoblasts of the external reproductive append ages (fig. 7, D, ill,.), located ventrally in Segments XI and XII in the female larva, and in Segment XII in the male larva. The imag inal cells of the mid-intestine are located between the epithelial e'ells near the basal membrane. (Fig. 7,0, ic.) The imaginal cells of the anterior intestine are located at the distal extremity of the (':iophagus, /!enerally known as the l'sophageal yalve (esv). The 2-4081-31--3 18 TEOHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE hypodermal imaginal cells are noticed in abundance in- late fourth stage larvae around the spiracles in each segment, from which point they spread, sweeping off the old hypodermis as transformation takes place. The imaginal Malpighian tube buds are located on the posterior extremity of the ileo-colon. ap B x F FIGUnE 7,-Lnr\-al parts and reprOflucth-e organs of ;J["crOCClltrtl8 gi{ucl!s£S: A, SpirnrJe of a fourth'stnge lllrvn with pnrt of ejlithelillm remo\'ed; B, Impression in hnrflcned silk of the int~rior of the salivnry gl:llld openin;;; a Is the exterior thread, 11 Is Il portion necumlliatt'd during the hnrdening pl'oeess, c Is the clillitlin;; liue at the point whet(' th(, two threads come together, and the shadrd areas on each side arc the Imprints of the presses; C, median longitudinal section throu;;h nnterior end of mid-Intestine of Courth-sta;;e Im'\'1l to show esophageal ml\'e; D. Jon;;itudinal sec tion through posterior end of hody "f Jur\'a slightly to left of medhln line; E, grosso modo view of mnle Ilnd .female rC'pro:luctlve organs In the larva; F, section through gonad of male lan'a The tracheal system is essentially similar to that described and figured in the first stage. (Fig. 4, D.) It differs from the first stage in that there are present nine pairs of open spiracles (fig. 7, A) situated laterally and slightly dorsally on the anterior margins of MACROCENTRUS GIFUENSIS, A POLYEMBRYONIC PARASITE 19 segments IT and IV to XI inclusive, connected with the lateral tracheal trunks by a spiracular branch. The lateral trunks are greater in diameter in proportion to the terminal tracheae than are those of the first stage. In each segment the lateral trunks give off branches which ramlfy to the fat, hypodermis, and other internal organs. The head is supplied from branches arising in the first thoracic segment, while in the last abdominal segment branches are sent out from both trunks and the short transverse ventral commis sure to supply the rectum, muscles, and the other organs and cells of the segment. st spr esp 14 LIl PWUltll S.-Internal orgaus lIud head pnrts of Illr\'llc of MacrOccIltrfl8 {/ifuc".~i8: A, ship "jew of fourth-stuge lar"a, showing Internal organs; B, section through sulivury duct; C, sections through snlivDI'Y duct and spinneret; D, spinneret seen fWIIl "bove ; J.J, horizontal sectiou through front pnrt ot hend nt leyel of spinneret; l~, lIIuscul"lUre of lwnd of fourth-stage lnrvu, seen frOIll uht"'e NERVOUS SYSTEM The nen'ous system (fig. 8, A) is of the usual hymenopterous type 10 consisting of brain, subesophageal ganglion, and ventral chain. The ventral chain extends to segment XI; the two cords con necting the ventral ganglia arf'. not fused as in certain Chalcididae but are quite distinctly separated. The ventral chain is composed of the subesophageal ganglion and 11 other distinct ganglia, the 10 Voulmsso\-!tch (31) has written a \'pry good nccount of the hlology and larval nnatomy ot ,If. abtlomillalis, a pnraslte of [>811111",0Ii8 hyulllwlis. In his drnwlllgs of the fuUy de \'eloped larva YOUkllBSQvitch Ogurrs two yolullliuc.us growths appearing 011 the posterior end of the nerve phalli. Th('se orglills lire In l'cnlIty the hlstoblasts of the reproductive npPI'I)(Hlgcs which IIrc suppllM with nerYl' branches from the termill,,1 gnngllQn. 20 l'BC.lHTICAL BU[,LEl'IN 230, U. S. DEPT. OF AGRICULTURE last one. of which probnbly represents two ganglia that fused since the first st,ag(', as there arc two pairs of nerve branches given off from it. The s0-caned Rympathetic nenOUR system is represented by a sman spherical fronta.l ganglion (fig. 5, E, Ig) situnted above the esopha gus just in fro.nt of the brain. The writer hils not Qhscn'ed the corpora a lIata. , ! IliGESTlyg SYSTE:'L 'I'he lli(resti\"(J tract (fig. 8, A) is composed of the mouth, esophageal pump, ()~ phllrynx, esophagus, mid-intestine, hin<1-l.11testine (which is divided into b'l'o distinct portions, ileo-colon and reetum), sali,oary glands and Malpjghian tubes. The mouth (fig. G. E), phal'ynx (figs. 6, E, 8, F, ('8]) nn larereb eloncrate~ cells with elonerateM and more or less irrerrularb nucll'i and many globules. From these ceUs as aIt'eady described comes tIlt' pel'itrophic membrane which incloses all of the undigested food and fills three-fourths or more of the mid-intestine; cnudad of this gL'OUP of cells is the mid-intestinal walt proper (mitep). This waU is C'omposed of an ontOl' layer of muscle fibers~ a basal membrane, allli n. lay('[' of large l'pithelinl ('ells with elongate irregulur shaped nuclei. containing granular and fibrolls matter and muny globules of un known natnl"P, llIost probably food material being absorbed. Be tween the celb of the wall oil the outer edge can be seen here and tlwre bunehes of small imaginal cells ('ic). In the posterior end of the mid-intestine the cdls of the wnIl are greater in length 11ear till' point where it joins the hinel-intestine. The lumen of the mid-intes tine is not continuous with that of the hind-intestine. In late fourth-stage larvae, however, these layers become less distinct at that point; the walls of the two intestines fuse but leave the lumen bloclwd by fibrous and granular mattl'r until defeeation takes place. l\L\(':1l0Cl~N~'ItUS GlFl'ENSI8, A l'O[,YE:MDIlYONIC PARASITE 21 The hind-intcstinc is di~tin('t1y divided into two portions, the ileo colon 01' anteriol' pOltion and the r~ctum or hindcL' PIll't. The ileo colon, whieh anteriorly is a short JlllrroW tube, thickens abruptly into a mOI'e or less conical clIla.rgement into which the two Mlllpighilln tubes are insert('d. (Fi~. 8, A, il.) The extreme posterior part of this enhtr~(,lllcllt \.)(,:II'S the imaginal buds of. the adult MalpighillI1 tubes (figs. 7, D, 8, A, imt) (bc:f.ore the larvn. hilS spun these tubes actually ~row to a. len~th of It millimeter or more). Immediately clIudad of this point is a mad,cd ('onstriction, which sepurntes the ilpo-('olon from the 1'('ctUlll ('I'). This ol'gnn has thick walls composed of cl'lIs with small round nuclei (fig. G. A, Iti) (not unlike the other n'lh; of the hind-intestine) and a wide slitlike IUlHen continuous with that of the iko-tolon. From this enlar~emel1t posteriorly to the Imus the n'etull1 is It straight though short ribbonlike tube {laUt'necl dorso\'entrally. The whole of the hiIH]-intcstine is ('o\'crl'c1 exteriol'l), bv lavers 01' llIusele fibcl.'s. 'Tile sali\'Ul'.\' glands (fig. 8, A, 8(J) consist of foul' dorsally bl'HnelH'd longitudinal tubl's. tWI) 011 each side of the mid-intestine, l'xtNll1 i ng 1'1'0111 tli" pO!-itel'ior ext!'l'mity of t his organ to the thol'llx. The tui.Jcs on one sid(' ('(lllle togetIl('r in tlw thorllx, £1'0111 which point a single tube ('(lntinUl':-l forward \'entrally to the head, wlll're it is joinl'cl just bl'lo\\' the mouth by the OLH' from the o(hcr sidc. Thl' (:0111111011 duet is a shol,t tuLc, U-shaped in cross section, extending 11J)\\'llrd to tIll' ('xh'rnal OIH'ning at the apex of the labium. CE'ig. 8, B, sel, C, 0.) The spinncret (fig. 8, E, 8]>1') lies in the opening of the sali\'lu',Y gland on the inferior lip, It is an elongate-oval passage fOl'll\(ld by two chitinolls prongs which Ilre outgrowths of the lowcr labial lip. Thl' superior lip of the opening is shaped like all in \'(\rt<,(l V, alld the ('uticle on ('nch side of till' V is considerably thickened, thus forming two " pl'csses" (fig, 8, C, D, E, 7)1') which, whcn pulled into positioIl against the spinllC'ret, oLlige the silk to pass out through this organ. The post('l'ior wali of the salivary duct is imprcss('(i by a. veltical IIwdiall I!roo\"c, The cells sUl'l'oLUHl ing thc duct whidl se('n'to the chitinous lining are similar to and continuolls with UH' h,Ypotll'l'lluti cdls, Immediately posterior to the point whc1'e the two duets s('pamtt'. howe\,('I', the cclls become di1re1' (;ntilltNl into IIlI'gl' !-ieC'l'ctory (,PUs (fig. 8, 13, 8(J) with oblong nuclei or considerable size (fig, 5, E, 8(1), :lIlcl this type of cell continlll's to the distal ('xll'('llIily of till' glands, Tlw Malpighian tllh~'s (fig!;. 7, D, 8. A, mt) arc simple tubl's COIl1 po~ed of large c('lIs with "Ollllll 01' oval l1udci and !l central lumen which opens illto lh(' hind-intestine. TIH'ir position is the samc Ill'; in the first f't:ag(', Tho Icgun1l'ntary Il1llscles include thosc of the head Ilnd those of the bOlly, Those of the hend arc thc two pairs of mOllth Il1llscles (fi~. G, E, 3,4) (,xtcncling £rom the dOl'sal wall ot~ tIll' mOllth to the cranium abovc it; the eight dorsal pharyngeal IIIlH.;cl ('S, thl'l'c pail'S l'Xit'IH!ing :from the pharynx dOI'f'ad of the l'l'Uniull1 (figs. G, E, 8, F, 5, G, j) and one pUll' attacitcd Illternlly but cxtcnc11Ilg llorslld :1Ilc1 sli~htly Ilnil'l'iorly to the craniulll (fi~s. G, E, 8, F, 8) ; Ilnd It ventrnl pllll' (fig. G. E, Hi) (>xt('!l(1 ing ventl'ally nnd ::;1 ightly postcrio!'ly from 22 TEOHNIOAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE the lower phnryngeal wall to the tentorium. Those of the salivary duct are, one pair of short muscles situated anteriorly to the duct under the spinneret (figs. 6, E, 8, F, 1), and one pair attached to the posterior wall of the duct (figs. 6, E, 8, F, 2) and extending pos teriorly and somewhat laterally to the lower wall of the pharynx. One pair of ruther fruil muscles extends from the tentorium posteri ol.'ly to the C'sophagus, (Fig. 6, E, 10.) rrhe maxillary muscles are attachNl posteriorly to the tentorium and extencl obliquely forward laterally and somewhat ventrally to the Il1t1xillary suture. (Fig. 8, Ii', 9.) The 1abinl muscles attach laterally and somewhat ven trally nC'ar the posterior border of the head and extend anteriorly and inward, attaching to the labial suture. (Fig. 8, F, 11.) The mandibulat' muscles, the most. prominent and voluminous of the head mnscles, attach laterally to the head wall, almost filling the cheeks, and converge inwllrdly, attaching to the cartilages on the mandibles. One group, the ('xtensors (fig. 8, F, 18), is not so voluminons as the other gI"OUP, the Hexol's (fig. 8, F, 14). The body muscle::; tU'{' al:i {ollows (Iig. G, B) (described from a preparation of the right half of the body): Segment I is provided with a dorsolateral group of six (dl6-1) (md a \'(lntrol,ateral group of f01l1' longitudinal (1.,7 4-1) muscles; th 1'('e isolated muscles extending obliquely in 11 dor~oventl'tll diredinn (dvo 1-3) ; two isolated muscles extending obliqllely in :1, ventrodorsal direction (?}do 1-2) ; the above muscles (~xt<'l1(l it'om the posterior to the anterior borcler of the seg ment; in addition there is a ShOI't transverse muscle extending along the inters('gmentni line (t 1), and another in the anterior quarter of the segment (t G).u Segment II is similar except that the ventro dor:-;itl 2 is nb,:ent; Segment III is similar to II; Segment IV (first abdominal) is similar to II with the addition of four other short trans\'(,l'se muscles (t 2-5) and two ventrodorsal oblique (vdo 34) ; Segments V to Xl arc similar to Segment IV, while XII has only the dorsolateral and ventrolateral groups, one of which extends from the anterior border of the segment to the rectum. CIRCULATORY SYSTEM The henrt is a muscular tubelike organ with a number of seg mental intake valv('s not unlike the typical hymenopterous heart as des(,l'ibed by Nelson (16) for the honeybee. It originates in the twelfth abdominal segment and extends anteriorly. The aorta, a ~mall straight tube, conducts the blood forward to the brain; the heart and aorta constitute the dorsal vessel and this organ is sus pended from the body wall by tiny fibers. The heart propel' is sur rounded by pericardial cells. (Fig. u, C, pc.) It is operated by groups 0-£ small muscular fibet's extending laterally to the inter segmental lines where they are attached, nnd muscular fibers within its 0\\11 walls; the pericardial cells lie between two layers of these muscles, thus forming what is cuJled the dorsal diaphragm. ItEl'ltOllUCTlVE ORGANS The reproductive or~ans of the 111:11e larva (fig. 7, E, F) consist of two small oval gonads located in the posterior part of the visceral n't'l)l'se muscles arc /lumbcrrll to conform with a sch!'lIIe already elaborated for other ftpecleB In earlier publlcntlollB (19), MAUROOENTRUS GIFUENSIS, A POLYEMBRYONIC PARASITE 23 cavity and attached by a short stalk to the center of rthe simple histoblast located on the floor of the abdomen at the posterior edge of the ninth abdominal segment. Those of the female larva (fig. 7, D, E) consist of two small ovaries in the seventh abdominal seg ment attached by short stalks to the small imaginn,l disks located on the floor of this segment. In addition there are the histoblasts of the ovipositor and sheath located on the floor at the posterior edge of the eighth and anterior edge of the ninth abdominal segments. ~'Nl' BODY ANI) UItNrE CELLS The fat body (fig. 6, A, f) consists o.f foUl' principal lobes extend ing longitudinally between the stomach and the layer of tegumen tary musdes as well as small pieces between the hypodermis nIHl muscu lar laver. The urate cells (fig, 6, A, 'lb) am interspersed among the fat cells in those portions of the four principalloiJcs which arc in contaet with the mkl intestine. Th('t,C' are no urate cells ill the thontx. ThC' oenocytes (lig, 6, A, 7, D, 0(,) nre found sin gly OJ' ill groups, princi pally laterally HettI.' the spiracles. THE PREPUPA AND PUPA FIGURE D,-Three cocoon colonies of .JlI/creaentr"8 The prepupa, and pllpa {JifuCIISi8. '.rhe sbriveled skin of the host may be , tl I 1 He,'n nttached to the lower hRlt of ench of the are 0;I Ie usua ly- two verticnl masses. X:!¥.! l1lC'llopterous type, The ~emale. pup!\. can easily be distinguished by the long ovipositor which IS curved dorsally and extends almost to the head. THE COCOON The silk spun by the larv!\. is at first of a delicate, fluffy, cottony texture and retains this appelll'llI1ce until the cocoons are almo.st completed, when each co.coon takes o.n a glistening finish due to. its glossy, compact inner texture. Each cocoon bears internally at the anterior end a small thick disk o.f hardened material forming a ro.und cap at this point. The whole mass is of a light-brown color thro.ugh which the larva, and later its dark-brown meco.nium, can be seen. The individual parasite larvae which have developed within a single host, upon emergence fro.m it, co.nstruct their cocoons gre gariously in un elongate mass, with their lo.ngitudinal axes nearly paraUel. The whole colony forms a cigar-shaped mass abo.ut 23 mm. long by 4 to 5 mill. in diameter, to which is attached the shriveled skin of the host. (Fig. 9.) 24 TECHNICAL In'LLETIN 230, U. S. DEPT. OF AGRICULTURE' BIOLOGY EMERGENCE When ready to emerge, the adult by its contortions splits the pupal skin inclosing it, works it off the body in pieces, chews off the small disklikc cap on the anterior end of the cocoon, and shoves its way through the remaining threads of silk inclosing the cocoon mass. Emergence may take place at any time of day or night. Usually the individuals of It single colony issue more or less simultaneously, although in some cases emergence may extend oyer a period of about 24 hours. LIGHT REACTIONS The adults are positively phototropic but not intensely so. The degree of this positive phototropism is such that it is a help in manipulating the adults 111 laboratory breeding and rearing work. FEEDING The adults of both ~exe::; readily absorb plain water or water sweetened with sugar or honey. The best food is dilute fresh honey water which they eat in such quantity that their abdomens become distended. They partake of concentrated sugars but slightly, how ever. Pure water prolongs their lives somewhat. OOGENESIS At emergence there is at least one fully developed egg in the lower part of each ovarian tube; sometimes there Ilre two, and occasionally three, thus nllowing for Il total of 24 eggs or more at the time of emergence. Immediately above the developed egg or eggs in the maturation chambers there are from one to three oocytes in differ ent stages of development, the most advanced being, naturally, the ()Be nearest the lower extremity of the tube; the nurse cells are obs(ll'vecl to be pouring their contents into the oocytes. Each oocyte has It large conspicuous nucleus surrounded by granulnr matter, which is in tum inclosed in a membrane of cellular structure with prominent, easily stained. Iluclei. The terminal chamber is filled with nl\l'se cells and germ cells in the process of division and differentiation. Females kept at a tempel'llture of 25° C. without moisture and fed on sugar water for three days were observed to have two or three developed eggs in each ovarian tube, thus augmenting the original number present at emergence by at least 24 eggs in three days. In two cllses one egg was found down in the paired oviducts nfter two days, and in one case three eg~s, but in other eXILminations after a longer period no eggs were tound down in these ducts. Females 15 days old have from 5 to 8 developed eggs in each ovarian tube, thus allowing for a total of about 200 eggs. Counts of eggs and oocytes show that a female is capable of developing at least 200 to 300 eggs. COPULATION The males are sexually active immediately after emergence and when they an~ placed with females mating takes place immediately. l\[ACnOCl~NTRUS GIFUENSIS, A POLYEl\[BRYONIO PARASITE 25 The female struggles to prevent copulation but the male llsually overcomes this resistance. No time is lost on preliminary ma neuvers; the male mOllnts upon the female, bends the tip of the ubdomen ullller that of the female and fertilizes her, the contact lasting but It ft"'" seconds. During this time the femu Ie may remain mom or less still or she may walk Ilround the cage dragging the male with her. In this case the male retains his hold only by the abdominal claspers while struggling to bl'llce his feet on the surface of the ('age. The female does not usually permit a second mating; hm' stl'llggles to prevent it are violent und consistent, though some times the male overcomes this resistance and a, second mating takes place. Sexual Ilctivity of the males is more pronounced when they lll'e not confined in a smull space. If mules und females emerge together in It small vial II. limited activity is manifested by the males, but when this lot is released into 11 lurger container the males again sturt mating. 'l'his is true ulso of It great number of other pal'llsitic insl'cts. It is probable that in 11 small space the air becomes that· Ollghly saturated with the odor of the females and the males who become satiated with this scent are less responsive to it as a muting stimulus. Tit is factor and the crowding which takes place in a small space render mating dillieult in ease th('re are It number of indi viduals present. OVIPOSITION THE FElIfALE DURING OVIPOSITION Females arc not ready to oviposit immediately after emergence but require a period of three or four days to develop their interest in the host, an intere!'it increasing as the number of eggs in the ovaries augments; by the fOl\l'th day interest is lively, and by the C'ighth day it is intense. Oviposition takes place best at a temperature of about 22° to 25° C. 'rhe females show little or no interest in host larvae when the temJ?cratllre drops below 20°. ActiVlties nre decidedly more bl'isk in the morning before the daily feeding. Aeer fpeeling the femnJes are somewhat sluggish and their inh'I'est wanes. Bright sunlight seems to stimulate ovi position activities. Females couhl~ot be induced to oviposit in Pymusta eggs. ·When supplied with host larvae in cells 01' webs on leaves of Rumex, Artemisia, corn, etc., the female immediately directs her attention to the infested point. The first indication of the ovipo sition stimulus is the slight unsheathing of the ovipositor. It is probable that this movement brings an egg down fl'om the oviduct to It position of r('adiness in the vagina or proximal end of the ovi positor. This supposition is strengthened by the obsel'vation that t.he females have :t tendency, once the ovipositor is unsheathed in this manner. not to sheath it again until an egg has been laid. l\. few seconi1s after this movement the female raises the end of the nbclollll'll, brings the tip of the ovipositor forward to a point under the body, and conunences to prod rapidly the area where the larva has been feeding. The antennae am extended forward and some what separated, the wings are mised, the ovipositor is helel pointing lonvnrd and downward. the sheath describes a loop upwards Ilnd ~"'IO~l-:n-.! 26 TECHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE bMkwards, curving around the end of the abdomen up.til it points forward and downward. (Fig. 10.) The sheath retains the ovi positor in its tip at a point about one-third to one-half of the dis tance from the base and seems to direct that organ in the forward and sidewise exploration thrusts. 'Whether or not the sheath actl1 ally plays any considerable part in the guiding of the ovipositor is soml3what doubtful, as it probably is pulled 111to this position by the ovipositor and retains a hold on the latter as the point of this instrument is brought forward between the front legs by the group of strong muscles attached to its base. This is the only position the sheath can assume unless it releases the ovipositor entirely, in which case it straightens out and points upwards posteriorly; this actually happens quite often, but the work of exploring and stinging the host larvae continues just as well without the aid of the sheath. It is noted, however, that the ovipositor can be bent slightly in any direction at its middle or near its tip by the action of the muscles of the sheath. 'When the female perceives that a larva is actually pres ent she becomes excited and her thrusts become more frequent; if the point of the ovipositor comes into con tact with the larva it is thrust into the latter, an egg is quickly laid, and the ovi positor is almost immediate ly withdrawn, straightened out, and sheathed. l!'loullm lO.-Fcmnle MaCI'occlltrus oi1"ensls in Because of the manner in position for ovlpositlng. X G which she manipulates her long ovipositor it is practically impossible for a female to sting a free-crawling larva. The larva must be in a web or a hole whence its escape will be retarded for the brief period of time necessary to allow the female to locate and sting it. The places most explored and apparently the most agreeable for the female to give her thrusts are under the edges of a leaf where the leaf is held flat against another surface and in holes in the stems and leaves, etc. If larvae are free on the surface of a leaf and the spot is designated by frass or a bit of web, the female will go through her typical exploration movements for hours and often not hit a single larva, especialy if the larvae are small (first or sec ond stage). The thrusts are made in any or all directIOns and an egg is laid only if by chance the ovipositor encounters the larva. During this time the point of the ovipositor may be in contact with the leaf in the vicinity of the larva, but it does not penetrate the leaf. Isolated larvae attract the female little or not at all. Occa sionally the female, thrusting vigorously to lodge the sting, will apparently "pursue" or "wrestle" with a larva driven from its MACROCENTRUS GIFUElITSIS, A POLYEMBRYONIC PARASITE 27 feeding point, but when such cases aTe closely examined it is oiten found that the larva is retreating and is dragging the female by a leg which it has seized in its mandibles. After givin~ a thrust the female withdraws her ovipositor, sheathes it, ana ~auses for an instant as if to repose after the labor of oviposition. ::;he does not, however, abandon the spot, but almost immediately resumes her exploratory thrupts, and if the larva is again encountered it receives another thrust. The larval burrow with its accumulation of frass and web has a very strong attraction for the female. Even though the larva leaves the liurrow, the female will remain in its vidnity for a considerable time (an hour or more), thrusting vigorously around anel into the empty burrow. No expression can better describe the actions and movements of the females than to say that they appe!tr to be extremely stU\pid. They CM not locate the host precisely and often stumble around over It larva without stinging it at all. Such behavior among insects is common, however, the rule rather than the exception, and numerous similar cases can be cited. IJundie (14) has observed that in the case of ApheUnu8 'l1Ulli Hald., when this parasite turns and thrusts backwards (as is its custom) to ovi posit in E'l'io80ma lanige1'wJn Hausm., if the aphid moves awa.y the pa.rasite remaills there for some moments thrusting into the air. The writer has observed an identical proceeding in the case of (Ohalcis) BmchY'l11e7-ia fonjJcololl~oei Duf. when depositing in larvae of species of Sarcophaga. Another case .analogous to these has been observed by the writer in mating adults of E1llimineria c1Y1,lJsifemur Thoms. (another parasite of P. mtbilaZis). The male moves around the immobile female, fanning her with his wings during a prelimi nary courtship p ...riod; if the female moves away the male will nevertheless continue his fantastic gyrations for some time before he eventually discovers that his mate has walked away. THE HOST LARVA DURING OVIPOSITION The attacked larva generally shows its excitement by quick con tortions of the body and by retreating farther into its burrow. If the burrow is shallow and the larva can not retreat, it leaves the bur row at once and squirms away rapidly; if the burrow is of moderate depth but not so deep as to permit complete escape from the sting of the female, thus presenting ideal conditions for oviposi.tion, the lar ya will most certainly receive a number of additional thrusts; if the burrow is very deep the larva by retreating escapes completely from further attack for the moment at least. When touched by a Macrocentrus, the larva ma.kes vigorous ef forts to escape. These efforts are tripled after it has received a trust, and often it will rush out of its shallow burrow even when a female is standing at the opening thrusting vigorously into the in terior. This sudden rush usually dismays or upsets the female, and in ,a case of this sort the additional thrust given at this moment does not succeed. POSITION AND NUMBER OF EGGS IN HOST The eggs are lodged in almost any place in the boely. The writer has observed them free in the general boely cavity, in the epithelial 28 TEC13NICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE tissue of the mid-intestine, in fat, muscles, oenocytes, or stuck close to the hypodermal cells; sometimes (but rarely) they are laid be tween the hypodermal cells and the cuticle. It is possible that some are placed in the digesti,-e tract, but these would probably be evacu uteu. One of the phnses of the biology of ill. gifuensis which the writer has tried to clarify is the number of eggs laid in a host by a female at a single thrust of the ovipositor. The importance of this point clln be appreciated when it is considered in connection with the polyembryonic development of this species. Unfortunately in actual practice it is not easy to demonstrate this point as it would be with Il monembryonic species. The simple process of allowing a female to pierce a larva and then recovering the egg can not be relied upon for the reason that in most ('~ses no egg can oe found. While it is possible that the female gives some futile thrnsts, it is more prob able that the majority of her '~hrusts are successful and that the failure to find an egg to account for each thrust may be explained by the probability of the egg being lodged by chance in some organ of the host and not floating freely out into the dissecting solution.. It is more often the case that parasitic Hymenoptera lay one egg at a single thrust of the ovipositor. There are, however, some ex ceptions, such as Apantcles tho7nJJsoni Lyle, which deposits 10 to 20 eggs at a single thrust (8), and Platygaste1' Memalis Forbes, which deposits 4 to 8 (liZ). The writer concludes, after a long series of observations, that gen erally one egg is laid at a single thrust. It is possible, however, that in some cases two or three are laid but not more than this number. The following are some of the principal observations upon which the writer bases this conclusion: (1) Many larvae have been dis sected in physiological solutions immediately after being pierced by a female. In most cases no eggs were found therein, in some cases 1 egg was found, and in no case was more than 1 egg found after a single thrust. (2) Laryae haying received from 5 to 20 thrusts each were dissected in a physiological solution; 1 or 2 eggs were found in each of several different larvae, with a maximum of 3 in one larvlL which had received about 15 thrusts. (3) It may be sllid as an objection to this line of reasoning that should 11[. gifuensis deposit 10 to 20 eggs at a single thrust they might be lodged in a group within a muscle or other host organ. The following data seem to refute this, however: In one case the writer, by attaching the larva as described under the heading technic, caused it to be pierced more than 20 times by a female ~lacrocentrus. This larva was then fixed and sectiolled. A number of eggs were found in it, some free in the body cavity, others in the fat body within muscles, and 1 between the hypodermis and the integument, but they were generally single eggs. No large groups or batches of eggs were found together; occasionally there were 2 or 3 near each other with out their presenting, however, the appearance of having been laid together as would necessarily be the case if a large number had been deposited at a single thrust. (4) Th~ long-sustained interest • and continued series of oviposition thrusts observed on the part of a single gravid female is another argument in fayor of the single egg theory. ~ As has been stated, a female 'will continue to sting a caterpillar, pro,·ided it can not escape, during an hour or more. J\lACHOCENTUUS GU'UENSIS, A POLYE1\IBRYONIC PARASITE 29 COHsidering that a· female possesses a relatively small number of eggs in her oviduct alld that they ripen rather slowly, it seems more r£)usonable to Ruppose that she would soon deposit all the available eggs in a. fe\\r thrusts and then desist. SEGMENTATION OF THE EGG The C'arliest stage of segmentation observed by the writer was in an unstained egg of unknown age (but le8s than 5 hOllrs). The fol lowing is a description of this egg: Length 0.12 mm., width 0.048 mm. There are two cells with clearly defined walls and nuclei in the cen tral part of the egg, surrounded by the granular contents of the eO'O', and unother cell or nucleus of a granular nature but different fl·~~l the other t.wo at th£) lower end; this latter may ue the germ-cel1 determinunt. Another egg in about the same stage of segmentation is illustrated from tixed and stained material in Figure 2, B. Figure 11 A, repre sents an egg less than 5 hours (Jill in ",hi{"h there are four'larrre cells of equal importance and "., three cl'lls or masses of protoplasmic material of an entirely different Hature. It i,> probable that these three are the 9' polnr bodies which will later form the paranu deus, while the foUl" prn larger ones are blasto 1I1(,l'es. A.nother unstained (i:\ ('gg observed ill physio logical solution is illus ~ tt·ated in Figure 11. B. ode. l"IGl:ltE 11.-1'gl;s of MaCI'OCCllirIlS girucII8is in different aIld the following' de~ stnges of dC\'clopm~nt: A. Egg le~s than 5 hours old (BoniJl's); B, egg Jess tlllln 5 hours old. showing s("l'iption applies to it: complete segmentation; C. <'gg 9'1.. hours old. showing Length O.1251111ll., width differentiation Into gl'rmillntivc (or embryonic) cells. paranucl!'us. lind troplllllnnios; D. cell in the dividing O,05U llllll., age less than stnge (metaJlhase) ;i hOIIl'8; there are 14 ('dis, 2 smalkr ones, 10 micl'OJls .in diameter, and 12 l:trger ones, ~O r:1icl'ons in diameter; the nuclei are round and clear and each is surrounded by It thin layer of granular matter, none of which occurs inside the nucleus; there are 10 to 12 small round L~lobules of unknown material scattered her" and there in the egg but lying outside of the cells i 4: of these are in one end; at the opposite ('nd there is a Illass of dense granulru· material partly surrounding :tnd inclosing the cells of that end; in this material small globules ean be seen, and at one point there is a conglomeration of t.hese globules and a thickenillg of the granular material, resembling a nucleus; this granular substance may be the polltr-body material destined to furnish the paranucleus; it extt-nels nearly huH way down the egg on OIle side and about a third on the other side. Another er;g ;) hours old has 18 cells of two sizes (the smaller one beillg ubout huH as big as the others), but of the same sort, while still another egg;) hours 01<1 has about 32 cells about equal in diameter 30 TECHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE to the smaller ones in the IS-celled egg. This egg has conglomera tions of granular matter at each pole, and along one side, partially extending in between the blastomeres, but no definite cells or nuclei can be seen in this granular matter. Other eggs of various ages up to 9% hours have been observed, but in many of these eggs segmentation is less advanced than in those in Figure 11, n. Often only 2 to 6 cells are present. Figure 11, C, is drawn from an egg 9% hours old stained in methylene blue. There are 2 cells (polar bodies) in the lower end of the egg and 4 germina tive cells in the central part, the former being distinctly separated from the latter by a membrane or line of differentiation; the central cells are in an area of clear material while the remainder of the ooplasm is pushed outward to the periphery where it forms a rather thick wall. GROWTH AND DEVELOpnENT OF THE EGG The next stage in the development of the egg (fig. 12, A, B, C), as observed by the writer, presents a remarkable change from the pre vious sta~e. The pregerm varies in diameter from 0.016 to 0.027 mm.. is spherical in form, has a rather thick outer wall, a layer of smoot:} ('ranular matl'ri aI, the trophamnios (tro) l ill which is embedded t! far gm prn tro 9c 'gc tro l1fc. nfc B ~h ",~:*o---,,;.;;<-- ph4. IrUH;lth1 12.-Prt\g-tuhryon.ic cells lire in anllphllse. ll.-E:\rly pregertu with two l'lllbr)'{lIlit' ':l'l1s. C.-Early lJregertu with thr('e embryonic ceUs. D.-Pre germ with fOllr embryonic cells. ]~.-Prl'gcrm with fh'e t'U1bryonic celis. F. J'rC!(I'rm .in whi<'h the (,U1hryollic ceIls at'.. ll()t associated in a permin:ttl\'c center (probabl}- an aborted ,'omlition). G -Another pregertu in tnl' sallle condition sllrrolluci('d hy plmg.;cytes point in segmentation anel that each pseuelogerm represents an egg which for some unknown reason (not lack of fertilization) has not segmented in the normal manner, and consequently is without embryonic cells. 32 TEOHNIOAL BULLETIN 230, U. S. DEPT. OF AGHICULTUUE Tho dilforellce in the number of cells often observed in eggs only a fow hOllrs old and the llumuer of cells observed in the pregertTl could be explained by snpposing that some eggs segment more rap idly than othel's and furnish pregerms snch as those shown in Fig nfc ...--", (JIj," lro l .; A' " nc. / nfk-~ pro pm 1"II01"ItH 1:.1,·· 'C\'t'm" alill psclIdogel'lll!l of J[(lCI'OCN"."u-s flif.umsfJl, A',-Pll('udogcI'DJ h:,,·ill/o! II paJ"Hutu'll1llS, hut IlO ;.!'('l"lllinuth'(l e(·II~. H.-II:i(,llld();.a·I~11l with two pur:tllllclei. (',--T\\'o ;;p('oud:try 1!('t'IlIS (Ol'lll(",1 frum thl) division of u primllry J,;l'rlll, I), )lltn'H't' of division of thl'. p.u':tnllci('lIs ill procp~s of encIrcling thc i;ermilluth'(· c('ntl'r. E.-FlI.'tlll'r dll'lslon or the p:trll II lIci l'liS, lC,-Sl'colI(l!try /.:eml, shu\\'ln/.: ho\\' th.. p:u'ulIlJ('l('lIs, III till' trnpillllllllioS. h:t~ SlIl"'olllll\('d th,' g'prlllinnth·c ('PlltPI', ~rwo p:u'nnu('ld nrc t:;(l('11 dh'itliut;. G.-S(lcolldary germ with III rl!:c parnllllcll'i me 13, D, E, This possibility is further supported by the fact that the writer has obseryec1 eggs of less than 5 .hours of age having 13 to 30 cells and other eggs!) hOlll'S old havmg only 4 to G cells, Thlls it wOllld spcm that the egg in Figure 11, C, which is 9% hours old, is ilJ the first stllgps of transformation to It pregerm; the essential l\[ACrtOOENTrtl':) GmUENS1S, A l'OLYE:;\IBUYONIC l'AltASrrE 33 elemcnts of the pl'egl'I'1l1 urc pl't'sent, numely, two polar bodics which may unite to fOl'll1 the pUl'anucleus, the germinative center of foul' cells, anci the trophllmnios Ol' outer wall. It is pointed out that Sih'estri (25) has followed the curly stages of scgmentation of the cgg of Af;cniaspis fuscicollis pmysincola, a t polyernbl'yonic ('halcid, and shows thnt the prcger'm is quite similar to that produced ill 111, flifuCl1sis, The same author (128) has illus tTlited the eady stllges of: H11c'!/l'I'us ?JUl!!"i, which ure similar to those of A. fuscicollis. Tlw next stuge in the development of the pregerm is illllstl'llted by Figure 12, D, E. These prcgerl11S urc 63 days old and wcrc kept lit tl:'wperatlll'es VUl'yhlg daily from 20° to 240 C. They lU'C of the slime strllctmc 1Il'i those previously recorded, but have four or five gPl'minlltive cells. Ji'igure 12, G, l'('pl'esents II pregerm loose in the yistl:'rlll Cll "ity and surrounded by phagocytcs. It is e"idently llbnol'mal, pl'obnbly Jlllving censed de"l'iopment in :1, stage before the germinatin! ccl1~ become us~ocillted in a germinative cenk'I" This jlregerm might 1m\'e been derj yed from lin egg 'which underwent COll sillel'able segmentation during its early days, as the number of gt'l'minati \'(~ ('('lis js seen to be markedly grenter than .in the other pregerlll!:i. The gerlll in Figure 12, If, was also loose in the body elLVlty nnd is sUrl'ollnded by phagocyte!';. Ji'igul'e 12, E, is 11 typical t'xlllIlple of the pr('g('rm of this stage. Examples taken from fields Sl'ptemher 1 and estimated to have the same age are found to be in identical stuges. The nl1mbt~l' of tells in the g(·rminative center contiuues to increase by mitotic di"isioll to about 20 to 40. This stage, though differing il'om the pl'l'germ only in this point, is here called primary gerIn in Mder to distinguish it fl'OIll other stagcs. The pamnllcleus now divides by fission nnd the germinatin! center divides by a central constriction, forming two more or less spherical germinative centers, l'ach uccomj)anied by lL paranUclell1: cell. (lfig. 13, C.) Each daughter germ thus formed is sUIToUlHled by and contained within the trophlllnniotic material. . This phase repres('nts the first absolute mUltiplication of a pre l'mbl'Yo whieh the writeI' hit!:; observed, and it establishes poly embryony. The two daughteL' germs arising from this division are hel'e ('ulled sc('olldary gl'1'1I1S. Figure 13, C, is an example fr01l1 field eollect:('d material about October l. Following one of the secondary germs during its development it is spell thnt ill the Ilext stage (fig. 1:3, D, 11j, F) the parauuclear cell di"id('s by fipsioll (D), giving 2 paranuelei, and these in turn divide in lin ilTeguial' tnnnnt'l.' gi"ing rise to 4, 01' ;:; pamnuclei, which by I'elllainillg in the tl'ophalllnios automatica.lly come to cncil'cle the gel'lllinati,'e (,(,l1h'r (F'), In F two paranuclei nrc seen dividing while some 0 f the germinati\'l' (·(·lIs arc likewise di vid i ng' '1'h(~ gel'minnti,'e ('('l1tel' of the secondary gl'I'1l1 is now composed of 30 to 40 ('('lIs and is eOlllpietely SlIl'l'oulI .....~,.~ .... .f."":,,~ .... ~ eC. i'_-"--c'--tro FIGl'UE H.-i:(·J'IllS 1I1111 lII(lrnlll!' In J/{/Cro('rll/rU8 fJi/u/'I/,sis: .d, Secondnry germ dlvl!l· III); [0 form tWil t<'rti!!!"), !;prIllS: II, 1I 3·lIIorulll botly; C, lin Is()illtcu morula; D, twO llIuruhw st'parating The g!,'t'ms iu sllch parasite boclil's may not, however, remitin to geth('l' \Jut (II nc1er the mechanicu I influence of the surrounding organs ()f til(' host, slId\ a,; tl'a('heal tubes, muscles, or nerve fibers, together with the general tendency of the trophamnios to enlarge and dis sociatp) may l'OI1H' apn rt. In tbiB case such :l dissociation is accom panied b,Y a. :, nowim~-ill ,. proceS8 on the pa.rt of the paranuclei, these 1l10\'illg within the tl:ophamnio:; and corning between the germinative ('('Ilters in "ucll :L mlUlIlel' that each geminati\Tc center as it dissociates from the othl'I':-; is sUlTOUnlll'd by the trophamnios. (Fig. 14, D.) MACUOCEN1'ltUS GlFUENSIS, A POLYEMBRYONIC PARASITE 35 In the process of dcvclopment from pregerm it is not known exnctly how lUany times the di vision by fission takes plnce. It is probuble thut this phase of the activities vuries nccording to the physiological condition of the host, location of the germs within the host, etc. It is even very likely that some of the pregerms do not divide at all hut simply continue their growth in a re¥ular and simple manner to pL'Oduce n, single morula. (Fig. 15, B, C.) However, the writer has no absolute pl'oof of monembryony. In the writer's opinion, morulae may be produced at :my of several points alon~ the line of development, such as from the secondary germ (fig. 13, D, G) or from the tertiary germ. (Fig. 14, A.) FIGUlUl 15.-:"Ilol'ulnc in ,1[uCl'o('cllt"uB oifuCIISi8: A, A G-moru1a parasite hody; B, two isolnted morular in fat body; C, i$ul:llcd morula surrounded by host lIssuc Thus thc term" morula ~, as nscd here indicate~ a germinative cen ter of til(' typcs repl'escntrc1 in Figures 14, C, D, amI 15, A, C, in which no 1110rc fission will take place, i. c., each morula will produce only one lalT:1. FlHlel' tllC'sr conditions Figure 14, B, represents a 3 morula plll'asitc body. Fip:me 15, A, a 6-morula, parasite body, and all such bollies ha\'ing morc than one morula may be called poly morulae. Figurc 16 is a schematic al'l'tll1gement representing the manncr of ~'rowth amI division of the parasite from pl'egerm to morula tlc('ording to the wl'iter's idea. - In general it sC'cms that dissociation 01' the polymorula is the rule rather thall the ('xCC'ptiOIl, for in host larnll' containing atlvnncerl !-ltngl's it is obselTc The morula continues its g'rowth lllHl after l'ollsidt'rablc increase in size tl'lllH;forllls directly ll1to It sort of oval disk with reflexed bOl'ders, (Fig, 17, A,) This disk by prolongation and clU'lin~ be comes mol'c sl('lIder ulltil it has somewhat of a IH'Il111tode form, Segmcnts then heg-in to appeal' (fig, 17, E) and organs bccome difTer ('Minted, •.\.t th i", stag-l' thc tro phamnios has <1i111in i,.;hed ('onsilierably in t hiekne,;s and the para nudei al'e flattening out. Fig-Ill'l' 17. 13, C. shows th is 1II('murane in a dl'''l'nl'ratinfr con (I i lioll, ,., '" PSEl"DOGERMS PSl'\ldogerll1s (figs, 13~ A, B. 18, A, and pI. 1. C) are found in parasitized larvae 8i Illllltalleow:;ly with til{' pL'e"crm, l' It e y un dOI:i)tedly l'e})resent f!'actions of Il dissoci ated efrn' ",hith are not pro\'ided"'''' Wit'I1 geJ'lIll' native celb. 01' entire eg-gs which' for some unknown reason fail to St'grnent ill the regular mannel', SOllie of these pseudogerrm; are mor sds of lL'ophamnios "'hi('h have dissociated frolll parasite bodies rluring- their process of !!rowth and dissocia 'iiol1. In Fig-lire Hi, A, F'Ir)l"HF: IG.--Ht'liNHnti(' nrrallg'('JII(lllt of de'"(.llo(ling formf!: (If .lla!'rtI<'Ollru,~ J/i{r"'IIHi,. to illustrate th(, rnl'lho(\ (If for example, It piece rnultiplit'lltlon IIlId disso('I:ltI,on 0( !(l'rrninlltivc ~l'nf('rs: (fl'f/) 1, nrhnnry ~1~1'11l; 2, ~N'ol1daT'Y j!l'I'm: ;1~ [t'rUal'Y gl'rm; is shown whieh ·L qU:.II'tCl'llury J.;(·rm; ",0, mOl'utu ill, ('mbryo will cedainly brc:dc awa \' frol1l "the main body along the' lin(' X-X. and 1n Plate L A, a silll)le mOI'ula is shown with It pi('(:"e (fry) which will. probably break away, 'I'll(' ('Ol1stitllC'llts of the pselldow'rm arc tl'Oplwlllniotic matC'rial and paranuelt'i, N"o gl'l'lnil1atin~ c('lIs :11'(' pr('sC'llt as in the germs and monIlae. The psellllog('rms al'l~ of all siz('s lip to abollt :2 mm, in length. The smal1('I' OI1('S and SOI1H' of thl' lal'g('L' or1('S are splIel'ieal; most of the larger ones al'(' oblong-~ O\'al, 01' il'l'eg-Illar in olltliIll', Oth('r8. liS shown in Figlll'e 18, A, :u'e of grotesqlll' shapl'!". PLATE 1 PHOrQMICf~OGRAPHS SHOWING EMBRYOS, MORULAE, AND PSEUDOGERMS IN MACROCENTRUS GIFUENSIS \. l'hntllluit'n'f.!I'lph .. hO'\lJ1~:l :'>ml!ll' 1lI0fUi:lll111 l:ltll ..tnj.w or dpytlioplIll'nl ..;urrotlnt!('d hS hn~t \lrg:Jlb, Tlw Pll'(,,> ·h~.... It!lI.lI(ld 'ff} \\ 111 pr()hil"l~ b"(·lIIl1t· dt'uplwd to form n p:'('lIdoJ.!l'flll. The pU'l'(' P'"'fJ11l h;l.... :tlrtl.lIl~ hl't'flllIl'dl'LH'tll'd {Bollin·... ),!1)dwlIl,dllfll :Iud t'o.. trw ); H. P:-I'WIO.l.!l'rIlI flf troph,111l1l1llt w (r'\J.!IJu'nt hptwi'.'n htht org:lo,,; (~. a :!~lIlnntla p'uH.. ill' Ilody, ,) ;'~lIlorlllll Imra..:ilt· hoil)', :11111 ~I p"I'udoL!t'fm ... urrlJllndtld 11,\ hfJ .... t It...... Ul'... ; I>, n J,!T{"lth j1l1lar~l'd pholntllit'ro!!raph ..luI\\ 1llJ.t a .... £·(·tlon IIf t Iw li·lllorllta para"U(1 hod) Jl1lhtratPd HI I·"jg:urt· 1;,•.\. ICli. ('UI it'll.'; t'. (1IIIbr~lI. ft'. t'lIIhr)lJuH' (':I\It)~ f, (at; Ill. IIlIl"I'h': /ilm, ph'I)!IlC'),H'''-; pfll. ptlr·ltIIlP!l'US; jJ.l(glll, j IhPlldoL;I'rttJ. try. (r lJ!lIh'ul. Itu, lrol'h'HuHio... ; I, \ 'wtlflll • ~ }\fACnOCEXTRl'S GIFUENSIS, A POLYEMBRYONIC PARASITE 37 The ps('udog('rms seem to grow by the absorption of nourishing material from the host and the continued division of the paranuclei. They divide by fission thus producing many smaller pselidogerllls. The tl'ophamniotic fragments d('rived from the parasite bodies or true morulae in the process of dissociation seem also to grow and divide. ht .....·:.c'~:-----,l-1I'T}.;:j~e ---,'i-ftI-1- ec. Lro p;.~_prn ~__ ht pm ec. '~"Mf-l-- tro io'WtJHfl li,-EmlJryo8 in MacrOCclltru8 uifllcII8is: A. Eurly embryo In edge of fat hody; B. O'mbryn showing degeneration of the trophnmnlos; C, late embryo show Ing further dcgcn('rntlon of the troJlhnmnlo~; D. (our 'embryos den'loping In the same pnrn~i!(> h(]d~': E, pamsi!e body of three morulae nnd three embryos; J," late embryo So far as the ,n-itcr has been able to ascertain, no pselldogerms develop into real germs. They continue dissociation until near hatch ing time when large number's of small ones having fiye to seven para nuclei are present. They remain in the visceral cavity and are eventually cat('n hy the third and fourth stage larvae. 38 TIWHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE CYST FORMATION Certain polyembl'yonic chnlcids (iJ.geniaJJpis fuscicolUs, A. testa ceipes, etc" during their development cause a definite proliferation of host tissue about the developing eggs in such a manner that all the (Terms IIrisill(T from a sin1rIe e'rIT remain toO'ether in a sinlTle parasite ~oody. Such,"" is " not the''''' cuse with"",., Jlf. gifuenS'is.e There is no"" such pro lifOl'atioll of the host tissue to form It TlllClellted cyst. The pregerm does, notwithstnnding, determine a swelling of the nucleus of the rnt cell in which it is lodged and of the nuclei or adjoining cells as well. (Figs. 12, A, TI, C, 13, A.) As the genu grows the chorion is pushed out.ward until it coin eides with the wall of the fat ('ell; as this takes place the nucleus of this cell i::; pushed to the periphery, and in tum otheL' fnt and blood cellf; nl'(~ dis plnC'(\(1. Tlwir walls and. eontcnts thus form It sort of impI'ovised eyst of mesellchyma tow; matCl'ial around the gcrm or polygel'm. If the genu grows to FIOI'UIl 18,-LIlr\'IlC of .J[(/CI'OL'C1llrus Ill{lIt'tlllis In pl'ocl'aa a large parasite body ot: hntchlng; A, l'H{>u In cases where the larvae are fixed in the muscles or fat of the host the stretching-out process results in the anterior portion of the parasite larva being protruded while the posterior portion remains among the orga,ns or fat where the germ was originally lodged. The larva thus seems to be fixed in this position by the anal ex tremity, but in reality the whole body is inclosed by the membrane. 1Vhen hatching is completed the larva retains no further connection with the host organs but is free in the visceral cavity. DESTRUCTION OF GERMS AND LARVAE In the course of development some germs are found apparently dead and surrounded by phagocytes. Some of the smaller' germs in a polymorula do not develop, probably because of their being in hibited in some way by their close proximity to htrger germs. Some embryos in the polymorula are destroyed at the moment of hatching by the earlier unfolding larvae. If the larva unfolds in such a way that there is an embryo near its mandibles, that embryo is killed by the chewing of the unfolded larva. Often for unknown reasons (perhaps associated with mechanical injury or crowding) a single larva or a larva in a parasite body will die without unfolding. The number of larvae dying in thIS m:umet· is ~reater in hosts bearing a htrge number of individuals. The earlIest hatched larvae destroy, in their feeding activities, many gerllls of large size as well as many hatching and some fully hatched larvae. The data in Table 3, taken from dissections of 16 parasitized larvae made ill the spring of 1929, will shed some light on the de struction of supernumerary germs and larvae. In this connection it is pointed out (as discussed elsewhere) that the average size of the male colony is 24 individuals, that of the female· colony 16, and of mixed sexes 20.9. TABLE 3.~D(lta on the lJara.site content Of 16 larvae of Pyrausta 1tubilalis from the field in t1w 8]Jri1~u Of 1928 --~ ------Contonts ---- .. -- _._--- -,---,------...... ,------I Number of germs I I Nllmber o( Inn'ne Numilefo(srnnlI N1 um I}ero( Ll\rvn mnnlotic (mg· Inrgn mnni', I! No. mcnts or otic frng· ' (~) Fir,t stngo Second Third s~~~~d. p~etJdogerllls mcnts with (1) MI(I· (3) !--_ .. _.. stlll(e- stn~e- nnd with 2 to 5 lIuml1rous Smnll dlo I Large I .? pllrllnllclei ~~n~~1. siwd !... _~J Alive I De~ nllve ~ :::;:~'!.. L .•• __ 10•• __ ...... __ , ...... 1 :10 20 ' ;; __ • __ ...... _...____ .._ ...___ •. ____ •__ _ '3.!:-.'••• ·._ .~__ r~\.I(II}O· .. '.'.'•• :_":'•• ~ ':.--.::~~ ...... ',: 35 25 ,~ __ ...... 1~~ .. _____ ----~ .. ~~ ------.-~--.------______7 2 a i- _____ ~ _____ M_~~ ____ • __ • ______.... ______ I-~: ~~i": ~ -:-~ ~ I ;[~~::- i:m ~':: :::;~ ::::~:i f: i-----~ :mm: --::: ~~~I-~:--~~ 1t::= ig~=:=::::::::::: :~~:=~:~:.·.:t:::::: "'''~f ::::::::: ~l !I J~ J?' :::::::: :::::::: 1:1.,••. aoo to §lIO.___ ._•• ;';,'\'('r"I ..i...... _. __ ...... ' 51 ..___... 70 Ii; ....__ __ lL::: ~~ i;, ~2~::-::::: ~::Jg .. ::1:::::::: :::::::: ::::::::'::::::::.:::::::: :::::::: :::::::: :~ 10. __ •. 300 to flIKL ..____• _...do .•: • ______..__ ... --.-----l.------i..-..--. _.. __ .__ '111 I Thoso nrc Chlssificd approximntoly vccording to the size of tho germs: (1) Secondary germs or small morulae hnving fi to 100 germitllltive colis nnd 10 to 20 pornnuclei, (2) momlne having 100 to 200 germinative cells nnd 20 to ·10 pornlllwlci, nnd (3) lnrge morulae about to tmnsform to embryos or having already done so. I In Inrva" Nos. J.1, 15, 011(1 10 the parn.~ite larvae o( nny stnge or condition were counted. Most of them were in the !irst strge, but no nccurate scpnrntilln into stages or live and dend cntegories wns made. 40 T1WUNICAL BULfJE'rm !:lao, u. S. DEPT. OF AGRICULTURE These dissections were made at ilTegular intervals, by opening pura :,;itized larvae in physiological salt ~olution und scritping out their contents. The datu refN' only to the material so extl'llcted fl'om the l!u:vne and do not include gerins and pselldogerms lodged permanent Iyin the larval orgnns. In distinguishing live lurvae from dead ones account wus taken of a cCltuin amount of mechanicnlinjury incurred by the pUl'Ilsites dm'ing the process of removing them. Most of the dead ral'vae bore signs of mechanical injury, the greater part of which wus due to attnck by othel' lurvne IlncIel: the crowded conditions. It will be seen that us the season advances und larvae begin to hatch, the pselldogerms and smull trophamniotic fl'llgments become 11101'0 IIUIlIl'rOUS. GROWTH AND }'EEDING The first-stage 1Ill'\'!l does no feeding until its head has issued from the embryonic n1l'mbl'llne (the trophumnios). Shortly thereaftct· it begins to feed. ExnminatlOns of the stomachs of first-stage larvae >;lIow that the fooc1l1bsorbed is composed principally of fat globules.12 'rhe stomach contents are arranged in three layers. The innel'most layer (fig. 5, A, ,-ita), surrounded by the peritrophic membrane (pm), is composed of fat globules to be digested. The second rayer (ml), outside the peritrophic membrane, consists of gmnular chyle of a more 01' less uniform consistency without or with small vacuoles; this laym' is probably partially digested. The outer layer (ol), similar to the second in composition but somewhat thinner, prdbubly consists of the completely digested material from which absorptIOn takes pl~lce. 'rhe first-stage lal'\'ae, when abundant in a host, do some chewing upon other orga.ns, snch as the muscles; occasionally (but rarely) a morsel of solid food is observed in a late first-stage larva; such morsels are observed more frequently in second-stage larvae, along with trophamniotic fmgments and pseudogerms. The second stage, lueking mandibles, ('an only ingest fat globules und such material as has been" prepared" by the first-stage larvae. Third-stage larvae ingest the remaining material prepared by the first-stage larvae and probably do some gua'wing and scraping on the host ol'galls with their weak mandibles. Pseuclogerms and trophnmniotic tragmellts also constitute a part of the food of this :!tnge. 'With the completion of the third stage the larva is ready to mi grate to the exterior, where it becomes an ectophage. EMERGENCE AND EXTERNAL FEEDING The first step neCt'ssa ry to emerge from the host larva is to free the head from the third-stage skin. 'rhis takes place in the follow ing mannel': 'rhe capsule splits, owing to the pressure from within, nnd the head of the fourth-stage larva is lifted out of it. 'rhis cnp suit', togetiwr with the former skin, begins to slip backwards, thus freeing the anh'l'iol' portion of the body. 'rhe lal'v!t now starts the .. Fillk (5) Hlnt~H thnt ,1[' ullcylillO/,/£ (cells ulmost Clltlrclr upon .. Ill'C(ligestl'd fooll, t:onsisting iurgcly or lymph, body fluids, und fnt globulcs," 110 cites no evidence, how ever, to show t.hut body nulds nn(.1 lymph nl'e ingeste(1. 'l'he ubovc-mentioned mnterlals, i( nhHlJrbed, CUll not in uny mllnnel' whutso('ver be conHidcl'rd liS pl'cdigcsted, :MACnOCENTllUS GIFUENSIS, A POLYEMBRYONIC PARASITE 41 work of piercing an exit hole. It presses the mouth parts against the body wall of the host und by constant scraping with the mandibles it pierces a hole through which it thrusts its head. By continued body contortions the larva ~r:tdually squirms out, head first,13 through the 1101e it has made. TIns process requires about one hour, and during this time the third-stage skin slips farther back on the body. As the larva completes the process of emerging it reposes for a few minutes lying on its ventral side with the hindmost segments still in the hole from which it came, then raises the posterior end slightly and brin~s it completely out of the exit hole, drawing with it the larval Skill of the first, second, and third stages which olten adhere to it. The posterior end is agaill brought into contact with the host skin at 11 point slightly distant from the exit hole so that the parasite assumes It position with its middle almost directly over the exit hole~ having its shed skin or skins under the posterior half of its body and the middle und forward part of the body resting directly on the host skin. It i,; now in the fourth stage. In this position the parasite rests for 15 or 20 minutes, if not dis lurbed by emergii1g larvae. After this period of repose, the larva becomes restless and manifests this condition by dorsoventral move ments of :its head and thorax i shortly thereafter it bends its head under the thorax, pierces another hole in the host skin near the exit hole, and fixes its mouth parts to the host at this point. III some cases, by ,Yorking its body backwards, the larva applies its mouth to the exit hole~ but more often a new aperture is made. External feeding then begins. The lalTa sucks into its stomach the material remaining in the host's body and does not. unless disturbed, let go of its hold at the feeding puncture. If pulled a"'ay from this pomt, a string of mate rial about the size of a horsehair can be seen extending from the interior of the host body into the mouth of the parasite larvu. 'When pulled this string will come out of the host's body to a length of 1 or 2 centimeters and out of the parasite's mouth to the same length. It hardens rapidly upon eA-posure to the air. Micro scopic examination shows it to be composed of host organs such as tracheae, bits of muscles, salivary glands, 1\falpighian tubes, cast skins and fragments of earlier stage parasites, pseudogerms, and the protozoan Pe7'ezia 7Jymu.sta PaiL which lives in the host Mal pighian tubes, salivary glands, muscles, intestine walls, and oenocytes. The parasites issue frolll the host at any point from the first thoracic to the ninth abdominal segments, generally at a ventral or lateral point. but sometimes dorsally. They are oriented longitudi nally within the host larvae with their heads directed toward the head of the host. They lie during the external feeding period with their heads directed toward the caudal extremity of the host. After !'Ipinning, however, when ready to expel the meconium, the larvae have again reversed their position and lie with their heads toward that of the host. These reversals in orientation are not in themselves remarkable but are only the natural positions occupied as the larva goes through "l~ini{ (,;) Htntp8 that the lana of M. IlIlGjrlh'ora. issues with the caudal extremit~· ('merging first; the writer holds tbis 10 be improbable. 42 TEOHNIOAL BULLETIN 230, U. S.DEPT. OF AGRIOULTURE a series of mechanical movements. As the larva prepares to issue from thn host it turns its venter towards the skin of the host; as it comes through the exit hole it naturally moves in a forward ven tral direction and thus finds itself, when outside, lying with the venter on the host and, because it was orientated cephalad within the host, now has its head directed caudad. Had it lam originally with its head directed caudad it would have fed with its head cephalad. The peculiar feature of this phase of larval activity is the fact that the larvae are orientated cephalad inside the host before they reach the fourth stage, and this can possibly be explained in connection with the direction of circulttiion of the host's blood. The second process of reversal takes place during spinning. The posterior part of the cocoon is spun first, and as it nears completion the larva revel'ses its position in order to complete the anterior part of the cocoon. Thus this phenomenon is not comparable to the prepupal reversal of cCl-tain Drptera such as the Hessian fly (Phytopltaga destl'Uotor Say) within their puparia. 'Vhen larvae issue from a host which is not in a burrow they are not orientated in any particular direction but lie mostly at right angles to the axis of the host's body. After a short rest they squirm into the most convenient position, pierce a hole, and begin feeding. External feeding continues until the parnsites are satiated if there are only a few feeding on a single host, or until the host is com pletely emptied. Some lalTae have been observed to feed for an hour and a half and others for more than 24 hours, but these last were cases in which only a few larvae issued from the host. In a normal case, where 20 or more larvae are fe'~ding externally on a single host, the latter is usually emptied in two or three hours. It is not, however, absolutely necessary that the .larvae feed externally; if remoycd from the host immediately after emergence, they spin and transform. in the normal manner. It is remarked in this con nection that this sudden change in environment from a liquid me dium to the open air, not accompanied by a radical metamorphosis, is perhaps one of the most drastic changes occurring in the life of any animal. EFFECT OF PARASITE ON HOST LARVA The first-stage larval feeding and chewing activities have a cer tain effect on the host, although this is not apparent miless closely examined, when the damage to the internal organs, more especially the fat body, can be observed. This injury is not fatal, however, for in cases where parasites die early in the first stage their presence does not prevent the host from transforming to the pupal stage. The ~ writer has dissected apparently healthy chrysalids and found therein phagocytized first-stage Macrocentrus larvae. The effect of second-stage larval feeding is but slightly more ap parent; the host larvae seem weaker and do not move quite so rapidly as healthy ones. Host larvae containing third-stage parasites, though able to crawl, are distinctly less activ~ ~il&n nonparasitized host larvae. Examination of the internal organs at this stage shows that the fat has been completely consumed, and the fat lobes sur l'ounding the mid-intestine, as well as the strips lying along each side MAOROOENTRUS GIFUENSIS, A POLYE1rIBRYONIC PARASITE 43 of the dorsal vessel and the fragments between the tegumentary muscles and the body wall, resemble pieces of lacework, retaining only some of the damaged cell walls held together bv fiue tracheae. The muscles are sometimes attacked to such a degree that they are thin a~d emaciated and are not strong enough to contract the body walls m the normal manner. If there are but few parasites, the internal organs, except the fat body, are not damaged. By the time the parasite larvae have finished the third stage the host larva can no longer crawl. As the parasites begin to emerge for external feeding, signs of life in the host diminish to a point where only a slight contraction of the abdominal and anal prolegs upon stimulation indicates that life is not extinct. The remaining internal organs previously mentioned are rapidly consumed by the external-feeding fourth-stage larvae and nothing is left of the host but the exoskeleton. SPINNING AND PUPATION The larvae rest quietly (after feeding) for about 24 hours, de pending Oil the amount of food they have consumed after issuing, amI then start spinning. During the spinning the larva bends the anterior part of its body about in all (lirections, attaching the thread here and there. The silk which isslles is a single threau, but if pulled rapidly it slips from between the prongs of the spinneret and comes out a double thread. If the larvae are placed upon a flat surface when ready to spin they are unable to form their cocoons properly, but scatter the silk over the surface in an irregular manner. After such spinning most of the larnlC die before transformation to adult. In cases whel"(~ for some unlmown reason the silk hardens in the opening of the salivary duct (fig. 7, B), as often happens in scattered colpnies. the larvae nevertheless go through all the movements and contortions of spinning, including the pauses to attach the thread. WIH.'n they have continued this exercise for a considerable period of time they become quiescent as they do when inside the cocoon. Death follows for such individuals. Each larva spins its own cocoon, and the whole group is bound together in a more or less compact mass. Individual cocoons can, however, be removed from the outer part of the mass without disturbing the others. The cocoons are completed in about 48 hours, and when finished the larvae cease their movements and rest quietly while transfor mation begins. This becomes eviuent by the change in form of the larva; the head and thorax become somewhat distended, a slight constriction appears between the abdomen and the thorax, the eyes become risible. and the larva is unable to move except for a slight abdominal contortion. This is the prepupal stage common to all hymenopterous parasites and many other insects as well. Shortly thereafter the meconium is expelled and the change in shape be comes even more accentuated. The external appendages of the pupa soon become visible underneath the old larval skin. This skin then splits at the top ·of the head and slowly slips off posteriorly, revealing the white pupa with its perfect appendages. 44 'I'ECHNlCAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE AVERAGE SIZE OF COLONY AND PROPORTION OF SEXES In the writer's rearing experiments, as stated elsewhere, the co coons were conserved in vials for emergence of the adults. In order to procure data on several important points, such as the avera~e size of the colony, the proportion dying in the cocoons, and the proportion of the sexes, careful counts were made of the adults issuing in the laboratory from 297 colonies, in the order in which these colonies issued. Of this number. 97 colonies had one or 1110re Macrocentl'llS dead in the cocoons; these colonies were set aside for other studies. It was then possible to count accurately und determine the !;ex of all adults issuing from the remaining 200 colonies. Among tlwse 200 colonies, 71 gave exclllsivel~r males with an aver age of 24 per colony, i:i4 gave exclusiwly females with an :werage of 16.2 pel' ('olony, ~nd 75 gave a mixtlll'e of males and females with an avemge of 20.9 pel' colony. Among the 54 female colonies, ;:; were composed of 1 to 5 ilH1i viduals each. 8 w('r(\ eompO>ied of 6 to 10 indi,riduals each, 15 were composed of 11 to 15 indi,-iduals each, 12 were composed of 16 to 20 indiyidlluls each, (j wer(' composed of 21 to 2;) individuals each, :; were composed of 26 to ao individuals each. ancl :3 were composed of 31 to 35 individuals ea.ch. Among the 71 male ('olonies, 1 was composed of -:I: individuals, 3 were composed of 6 to 10 individuals each, (j were composed of 11 to 15 indi,'iduals eaeh, 16 were composed of 16 to 20 individuals each, 1~ were composed of 21 to 25 individuals each~ 21 were composed \d 26 to 30 individuals each, G were composed of 31 to 35 individuals paeh, 3 w('re composed of 36 to 40 individuals each: 2 were composed of H to 45 individuals each, and 1 was composecl of 4:i individuals. From the 75 colonies producing both s('xes, 840 males and 733 iemalesi>,sued. In order to make a more complete study of this material thC'se colonies have been arranged (Table 4) in two differ ('nt ways. In the left half they are arr-anged in the order of increas ing number of males, and in the right half in the order of increasing I1umber of females. These colonies ('an be further divided into thrpe groups, those in whi('h mnl('s pL'edominate, 41 in number; those in which females predominate, 31 in number; and those in which males and females are equal, 3 in number. These groupings, however, are not taken into aecollnt ill the arran~ell1ellt of the table. l\IACROCENTRUS GIFUENSIS, A l'OLYEMBRYONIC PARASITE 45 TABUl 4.-:\'umbcI" of male and female adltlt.~ i8.~uillg frmn 75 mixed brood colonies Of Macl"O('clltrus !lifll('lIl:1i.~ -----·-~-I Ascending ntllnber or Ascending number or males females ------1--,------I ~ 1 I ~llIles .Felllllies' 1'ollll I Mules Females TollIl '-:--1:: -I 21 2~ Il . 7 ' 1[, 1Ij a7 18 III :1 II :1II 1\ I a :J Iti Ii H a :I;) HI II 1:1 ·1 I ~. :I. :II I IS ;j~ " :11 a III s s "~1:1 S II l~ 17 II 14 1:\ IS I, :. !!H.,::;; Ii I ~~ l,j ~t X :! 7 Ii I~!I s :i 1:1 \1 II II j¥;1 I~ ;i IS "I; la II. ii II r. IH Ii I;; :!.I ", ~~) .o} I 11 Ii 1:1 ~;\ jl ~ Ii 7 II 21 11 1,1 I, 7 iii Il a Ii ti I • s s f\:1 l~ Il s s Ii s :\ .. Iii" :~ ~~ IS 7 1I .1 ~l :,.1 ~~ S 1I tft :!., S S \1 I.; 21:\ S S tI ~ III 1:1 ~\ II ::\ S II '! t:i .f 1.1 s Il a 1·1: lf 17 .s II s IU' Ii lJ II 12 \) II IIi :!:I27 .;",1' 1:tS U 12 7 HJ l!l (I I'! Ii) ~1 20 \) I!! II Zi ,i,',j 12 III 22 1:1. IIi :!.S HI 10 !..'H I:; .' IS II f) II 17 1:1 .< 21 ~l 12 11 2:l 1:1 \I ~2 5 I:.! 17 1:1 HI II 12 2:1 14 ;i la IS J.I 1:1 IU II 7 1:1 2() 1·1 I III 1:1 2:1 In IS la :11 If, :1 H 17 1;1 .. II 21 1 15 i Ii 15 21 I';. ~ 15 :!:I hi " 'I I,; 21 17 I. !I Iil :!4 17 .; :1 IIi I!! 17 Jr; It; 2a I~ 1 II 1'1 2i I.~ 1:1 I:! lti 2S lUI 11) H iii ao 21) :; 17 u; :13 ~u 17 !!2 ~u '.., t~ 22 21 I , ' IS 2H 22 1:1 III a:!. 2:{ I :!I 22 " .).) 31 .\ ali ~ 3il '''______SIf) ,'1;\ , ______I, :;;'j itc. ______'liO ---''-___ 46 TECHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE From Table 4 it is apparent that the size of a colony is not a .factor determined by the volume and mass of a single host larva considered ItS environment and as food supply. Consequently it must be determined by the potential power of multiplication under given conditions of the egg or eggs laid in the host. In other words, an egg can not pl'odllce as many individuals as a single host larva can successfully nourish, for if this were the case there would be but few (if :my) colonies smaBer than the maximum (47 for the male sex and 35 for the female sex). As has been pointed out under the heading of larval habits, many l:ll'vlle are destroyed in cases whel'e a single host has more than 40, some are destroyed when It host bellI'S 20 to 35 larvae of the para site, and a few Ilre destroyed when the colonies are less than 20. From these facts the legitimate conclusion is drawn that the number and sex of individuals issuing from a single colony do not represent the content of that colony when it was in the germ or early larval stage but represent what is left after the thinning-out process has been completed. Leiby, after careful study (11), has expressed the opinion that the origin of mixed broods in (/opi(/oso17la gelec1l:iae, () tl'uncatellum, Platygastel' vel'nalis, P. MenUln~, P. 'L'al'iabilis, and probably P. felti is explained by the depostion of two or more eggs in a single host. 'l'he writer is of the same opinion as regards M. gifuensis, as he has no evidence to the contrary. If such is the case, it is quite evident that the great majority of parasitized host larvae receive two (or more) eggs,H not necessarily from a single thrust of the ovipositor, however. Il'llI'ther examination of these figures, on the assumption that the 75 mixed colonies represent at least li:iO egg depositions, shows that in 83 of these cases there were produced the average number or less than the average number (10.45) of one sex not accompanied by 10.45 of the otlier sex (the average for mixed brood being 20.9). The writer concludes from the above examination, supplemented by other calculations and observations which are not included here, that the smaller colonies ((j to 10indivic1uals) represent more nearly the product of a sinrrle egg than do the huger ones. These conclu ~;i()ns nrc somewhat Eu·ther substantiated by observations on the size of the lllrg('st polygerms and on the lingering oviposition hllbits of the female i1l. gifllensis. PARTHENOGENESIS No complete data arc available to demonstrate parthenogenetie reproduction. In certain experiments, however, eggs from unfer tilized females luwe been observed in a more or less advanced stage of segn!ellt.ati~n. (Fig. 12, D, E.) This fact permits the supposition that tlus species can reproduce parthenogenetically. .. For If 75 Illryne out of !!OO r~celvecJ !! egg!!. 1 fectlJizecl Illlci 1 unfertilized. it Is e.'t. tremely ilkI'll' thut n portion of the remnlning 1!!5 received 2 fertilized or else 2 u,lI\'rtliiz('" ('<:I!'S ('iIeh. ~rhis holds tme (lilli' If inscminutloll Is II ~hllll(,C 11roc~ss and is not controlled by the femulc. • MAOROOENTRUS GlFUENSIS, A POLYEMBRYONIO PARASITE 47 CONSIDERATION OF THE ZONES IN WHICH MACROCENTRUS GIFUENSIS OCCURS PHYSICAL CHARACTERISTICS The Armorican zone, in which this parasite is most abundant, is a rather low region (altitude 48 meters) of rolling land, rich in pastures and cultivated lands. The principal crops are cereals, forage, and grapes. The soil, of Jurassic and Cretaceous origin, is mostly loam and day loam with abundant calcareo-siliceous outcroppings. l<'lGtiltFl l!l,~·-('lIl11oJ.:rnphs (01' threr zon'~ In which J1u('roccntl'us Ilifuel/sis Is known to "e,'lIr :IS II [lnrn~ll(! of 1'/11'<1118(1 1;,. .Ji/a/is, Letters In thu grnllhs stano for the 1I101llhH of III(' Yl'ar Artemisia, the host plant of P. nubilalis1 is not extremely abun dant. Occasional patches and isolated plants occur in the fields and lllon~ the roads, but it is mostly confined to waste weed areas near towns, along railroad tracks, river banks, canals, and garbage dumps, and it is in such places that P. nubilalis and 11/. U'ifuensis are found. '.rhe soil of the Cenb'al zone, in which this parasite and its host have been fOl1nd (St. Etienne and Limoges) 1 is of Presilurian and Carboniferous origin. The area is of rather limited agricultural 48 n:CHNlC.\L lIl'LLETIN 230, U. S. DEPT. OF AGRICULTUHE illlpodullel'. lHlving lllueh of what is described as " dead" or unpro ducti\'e soils. In the valleys, however, the soils are a11l1\'ial, often bluck frOllt the influene(' of mineral-bl'aring strata, and are Bome what productive. In othl'l' parts the soils 27'~~~~~~~~~~~~~~ i formed by the decom 26'~~~+-+-+-+-4-4--r-r~i-T--r position of schists and 25 sandstones to It consid 24 --5E~#UAl~Tto/ZO;VEp:-,e.?I\IC.Q erable depth support ------·SE9tt41\1.t4A1,.wOU~ZO/YEFe4;VC6 pastures and meadows, 23 ._.___ P4AM~..s1A/YtWO.RTto/.za¥Etf'u'f5J.i lImi when sufliciently 22'~-~.-~-r-r-r-t--I--r-r-Ti~~- tl'l'ated with lillie they will produce cerl'als. Che~tnut and other Jorests are present. The parasite and its host are found in situa tions !"irnilar to those of the Armoriean zone. The part of the Se quaninn zone (south) in whic'h this parasite has \)el'n found consists of the r'!lthpt, flat lands along the Seine in the vieillity of Paris (alti tude GO meters). The soil, of Tediary origin, is a fertile mariaceolls and ealcareous day soil. The pr'ineipal croi)s are trllck CI'OPS and frllits, including grapes, ~\.r tpmisia abounds in the alHlIHlant waste Ianels around the dumps, fac tories. and railroad yards: CLBIATIC CHARAC· TERISTICS W'ith the prl'sent lim ited knowledge of the distribution and hosts of i.ll. {lifu8l1Si8 it is .FIOI'HE ::!O.-Clilllographs for two zoncs In which .1[". impossible to discuss cru(:(llitrus yi(UfllHiH is known to OCt'ur ns a parasit~ uf l·Y"(/II.~t(/ IlIlbi/utis lind Olll! In which It does lIot the climatic cha!'aeter o~·(ltlr~ JL yifu(!'H~i.~ docs Hot OCl'Ur in the Sel)uaninn isti('s comprchen,.,in,ly, (north) ZOlll', I.('tters In thl' graphs stnnd for th(, rnnntht-; of th(' ytlllr The eli mOl-n'n phs (figs, 1!) and 20) for the Arlllorican, Ccntral, Sequanian, and Danaprisian zones. and for til\' regioll of KII1l10ll1oto (based Oil data from N agnsaki) ~ show tIll' g-t'Il(,l'Hl eharaeh'risties of the eIimates ill which 11£. gif'U"·I1Ri.~ oCCUI'.~, 1\IACTIOCENTTIUS GlFUEXSISJ A POLYEl\ SEASONAL HISTORY OF THE PARASITE As is demonstrated in Fig-ure 21, the seasonal history of the parnsite syncluonizes extremely well with thnt of the host in the ,Armorican zone, where it hns been intensively studied by the writer. The peak of adult emergence occurs during the.Iat.ter nart of .June and the majority of the eggs are deposited during the "first half of .Tulv. The se::rnll'ntation and dHPlopmpnt of the egg take placp ratller slowly. In the first half of September the tiny spherical pre germs (fig. 12 B, C) are abundant; as the autumn advances a con siderable number of primary and a few secondary germs nppear; to It wlll Ill' nl'Cl'ssnry to nwnlt the results ot studl('s now bcln~ mnde by 'V. B. Cart" \n'Jght I,,-(ore detlnitl' ('onciusloIlH cnn Ill' drawn In regnrd to Jl. {Jifllt'11His In. nreas having two or thr(ll.' gf~n('rHtI()ns. rnw uho\"p i~ U)('lltiOI:l'd us It l'ulllioll :I !!'uim.:t t l'rOlnOUS ('011 du~lonij Iikl'l~' to hI' IlInde wh"n tht! Europ"nn zones nlone nrc consldt'red. 50 TEOHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE these are the stuges most common during the wintel·. (Figs. 12, E. 13, D-G.) In eudy spring the growth and division are intensified lind pl'oceed rapidly. About Febl'uary 1 the parasites lire in the lute germ and smull morula stages. From this time on to the middle of March the smallel' secondary germs increase in size, while the tertiary genlls und smull morulae grow into embryos. During the latter Plllt of :Murch the germs in a host luna can he classed roughly into three more or less distinct groups.16 FIGl"1I1l 21.-Sellsonnl ncth'ltil'S o( Pl/raul/ta 1Iubi/flUs ,ulIl Its pllrnsltp. J[flCrIJC('Il' Irul/ I/i{IICIlHis, IlS dcll'rmlnc '" III tnhll's sdUn!; forlh ('xpl'rlnlt'ntnl tIntn thesn groups will be dcslgnnt"d by 11). I:!l. nll(1 \::). )[ACltOC~;NTHUS GIFUgNSIS, A POLYEl\IBRYONIC PAHASITE 51 ltu'ge secondary germs, 0,5 to 1 mm, in diameter, in which the em bryo is forming lind the ger'minutive cells are armnged in a slender cUl'led filament in the embryonic cavity (E), The majority of the genus are single, but many polymorlliae of from two to six germs llre present. Often, with the dissecting needles, it is possiblc to separate two germs which seem unitecl in II single pal'llsitI.' body; in othcr cases it is impossible, The writer be Iie\'l~s that in cases where germs are easily separuted in this manner tIll'S IIrc abollt to eome IIPIH't in the natlll'lli process of growth und lIi \'ision by fission. Germs 'In th(l slime pllrusite body IIrc in the Same 01' ulmost thc SUIlIC stage of deVl'lopmcnt. In Figllre 17, E, six germs al'e repre sen.tcd. t.hree of which nre in the early embryo stage and three of \\'11Ieh lu'e mOI'ulnl'. In IIddition to the above grollps of trlle germs, thcre are fOllnd in eyery parnsitizl'll lurnl at thc same time IUl'ge numbel's of pscudo W'I'lUS ()J' trophumlliotie fl'llgments (fig. 18, A), as described else whel'e. In a single parasitized larnl there may be more than a thom;Hnd of these t'l'llgnll'nts antI in most eases there nre severul' hlllllh'ed, The lurger pUl'Usite bodil's ure free within the body cuvity; the single morllla is found free 01' lodged in or between orguns such as fut eells 01' llIuseles. The majority of 1111 forl11s are found in the posteriol' part of the host's body. They probably are lodged ill this portion of the host beeallse of the blood flow. By the first of April hatehin/! ha!; started. The date of maximum o('em'I'('nee of liI'st-stll/!e lan:ae is about the lust of April. Growing lun'ae an' th('n present untIl the Intter part of )1ay, when cocoons bc/!in to aplwar. The maximum pupation oeClil'S ubout .June ij to 10. Duta from the Danaprisian (north) zone are somewhat mengel'. Host IUI'\"ae J'l'((li n'd at HYl'!'l'S in XOn'mlJer, H)28, gll\'e, by dissection, lute ~el'ms, ('ad,\' (lmbl·Y0"i. and first-stul!e lar\'ae of 11/. gifuelUris-a con(htiol1 eorresponding to the development of about April 1 in the Armorlean ZOIH'. It iH probable that. on account of the 10n(1 cold wintel's and thc more deeisi\'e and abrupt changes frOll. one se:~on to nnothel', tIll' development of J/. gif'Uell,~i,~ proceeds more rupidly in spl'inl! und Sllll1nll'r in that ZOIll' than it does in regions hu\'ing a more l'qullble dilllatl'. This might l'xplain the different seasonal l'hythll1 of the plll'Usite in this ZOIlI'. In .•Japan (Kulllomoto. )fiyuzuki. Shizuoka) Cat·twright has rea n't! this !iIWl'ies to the adlllt ;;tul!e from July to Xowmber. the majority ellll'r/!inl! in ,J Illy. a considenlble number durinl! the first half of AUl!ust, and seattel'inl! ('ol'onies hom that time until the last of October. It is probable that the high tl'lIlpel'Utlll'e ancl hllmidity pl'evailing in Japan dlll'inl! the SUIllIIH~r months al'e responsiblp f()r two (or more) l!enel'Utions pl'r yea 1'. EXPERIMENTAL DATA In thut portion of this bulletin which deals with experimental data the writer has sought to II \'oid h'Ill!tlw tables, Thel'e are. how ever, se\'eml phase;; of tlH'ill\'estil!ation~ whi('h ('an I)('st he pl'e ~ellh'd in tablllfll' forlll. 52 TECHNICAL 13l'LLETIN 230, U. S. DEPT. OF AGRICULTURE Although certain of these tables may seem to resemble others III some points, this i$ in reality not true, ns each one is presented in order to demonstrate It certain limited phase of the ach..·ities of 1.11. gifuensis. LABORATORY BREEDING EXPERIMENTS The ,,,,·iter conducted It series o"f experiments on the earlier ,.:tages of de\'elopment of the egg in order to determine the rapidity of development. In all, 420 host larvae were isolated and exposed to :,!l"avid fe1l1al('5 in th(, manner c1escribed under Technic (p. 6). Of the parnsitized lnrvae, some died, probably from superparasitism and :-;oll1e from unknown causes; It great number pupated before the para site egg had time to develop; and others, after a period of incuba tion, wel'c found to be hl It poor physiological condition I. with no live parasitcs present. 1Vhen sectioned, about a dozen larvae were founel to be parasitized. After 36 dt\,ys at 18° C. the eggs were found to be in the pregerm !'tage of development, und aftcr 43 days they were but slightly further advanced. At 22° C .. after 63 days, they were found to be in the stages repre ::cnted by Figure 13, D-G, some having two paranuclei with 6 to 10 gel'minative cells (primary germs), others having 8 to 10 paranuclei with a centcr of 20 to 30 germinlttive cells (large primary or second ary germs). 'Ill(' above data. arc taken from eggs bid by both fertilized and unfertilized females and coincide with stages of development found in la I'vae taken "from the field on August 29 and estimated to be about two months old. Xo colonies werc brought through to maturity from laboratory . ..- ········-·~-I:-;-llllllll'r· ~cr ('eDt -1---- Total host. larvlle in l'Xlwriml'Ilts ~ .. . .+~~~_.~ ...... _~ ... _._. __ ", __ .___ ~,.._. 1 3.24; HJ() J)i~d ...... ,. illS 23.fi Pupated••..•.. _____ ••....•..•.•. ..: ::: :::~:::::: ::~ ::::::::::::::i I, i88 1 ;r.4 Olive l'Oloni~s of ~r. gifueosis.•.. ~. k ~." ...... ~ __ ~ _____ ~ _____ ~ ____ .. __ ~ ... __ ~ ___ ~.1 (i!1l f l7 'J'hese Inn'lIe wl're characterized by several distinct phenomena. (1) The fat body was poorly developed. the fat cells and their nucleI beinll: ells!ly distinguished under low· 110Wl'r IIlllgnltlcation heclIllse of II lack of fnt glohules; (2) hundreds of small black spots w('re Jlr~sl'nt throughollt the fat body; (3) the fut usunlly surrounding the heart and reproductive organs WIIS I'ntlrely absent; (4) the pericnrdlal cells were enormously de· \·~~Ioped. The IlIrVlle werc nlso hCI1V!ly pnrllFltlzed bv the protozoan Perezla pvrausta l'all.. whIch WIIS prl's\'llt In the sllll\'lIr~' .donds. Mllhllghlan tuhcs. Willis or th~ mid· Inl.('Rtlne. nn.1 mn~r.ll's. It Sl'~ms to tbl) writer that these protozoa must ha,"c Interfered consh1"l'ohly with the mNubolic 1I('tivities of the host lu 1,\,11 ('. :\[ACUOCENTUUS GIFUENSIS, A POI"YE:l\IBRYONIC PARASITE 53 SUMMARY OF DURATION OF VARIOUS STAGES The approximate length of the various stages from the time of emergence of the parasite from the host larva to the time' of adult emergence is given in Table 5, and a summary of the longevity of adults when pl'Ovic1ed with food and moisture is given in Table G. Adults of both sexes, when left without liquids such as sweetened or plain water, died within two or three days. . 'fAIH,E J.-811111J1Wn! of appro:r:imate lelluth of various 8t(/UC,~ of Jlacrocclllru8 !/ifllellsis after emcr!ICllce frolll Ille host larva I From ('Ilh'r· gl'lll'(I from j Externlll s· it nin" Hesting 'l'j~IIlI1l'rutllrc i (l'('dlog '".1 1 t'> 1'111'111 host tn 1 stage lI stngl~ slilge slllge trnnsror , , mntiou to j IIdlllt ~,------.------1· J)IIVS J)IIV,' Dnvs ~j'.) (' _. I 1 S !!5° C I ~3, I 1(11 ~WO (' 2·~'; 13,~ ISO (' !U2 1~4 10I. I --.--.-~---- T.\m.~; 1l.-811J1Mllary of kngth of life Un cla1ls) Of adults Of jJ[(tcrocenlru.~ !lilllclI.~i.~ 1eifh {ood allll '/IIoi.~lul·c Indosllrl.\ IITlo'fIlPI'rn9 St'X" [MaXimumJlldivid· Avcrng:ll for Bsingi' tUrI' . • , uuls longe"iLy indi\"idllal !-~-~,~. - 'I N:mbe,· --~~;8-' .-;;:;: .. Lnhorotory _ 11;"2·' :'Inl,'s....._...... " 15.4 23 Do...... _..... IIj-~·' Ferti\lzetl remales...... _ 21 10.. 30 H.. rrigrrntor(couslauIL .....: 18._•••c1o ..___ ...... ___ 2. li.i 36 I>o .... ~. .' 18 l~n(('rtiIizcd retnnlcs'_' ___ ~"'1 111 13.8 ~_:Ol Do ,I .....do...... _ ' ...... 1 14 10.0 RAPIDITY OF SPRING DEVELOPMENT The following experiments were planned to show the influence of temperatlll'e and moisture upon the rapidity of spring develop ment of the parasite under diiferent conditions. The host larvae. containing pilrllsitc germs und embryos, were collected in the fielll and immechatcly pluced in incubators. Certuin boxes were kept dry and others were supplied nbundantly with contact moisture by plunging in water at regular intervals. 'rable 7 giyes the results of thes(' experiments. • 54 TECHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE T.HILEl 7.-RapidUy of .~pritl{J tlcl'eloplllellt of Macrocclltrll8 yi{lIclIsis 1IIIder (lit terant cOllliitiolls and synchroniza,tion of p(/.I·a,.~ita "/I/cta/JUlisl/l. H:itIL t!tnt of host 'lIIHZCr fa'vornblc cOllditiOIl.y Approximate i,' Average time stage or parasite rrom begin· development 1 'rem· Colo· : ning or ex per· Labo· DI~~.;,~I'I-----'--ll~~~8~t Mois. 1t::~:· Hosts Para· ~rg(nChrys. Iment to ratory from Oerms which ture bllslis died ~itisn~ fue'nsi~' alids --,-'- No. lleld ex in lot In lot de,·el. :formed Spin' , i ~:~. poS;d oped' ning ~~~~i (I) (2) I (3) o~o~~ : host 1929 I . -:;;- --;Number No. P. cr. Xo.' No. I~;'i DOllo 3008.... 1I111r. 2., .••• I' 3007.•..•_.tlo"...... I t :~ i~~~::: I~~ 6~ 18 ~ : o~ : ~~ : ~~ :w.,0.••• }Fcb. 8 10 3051.... 20 WeL.' lSI 27 ao ~i 107 : ~ii ; 39 30.,3,\.. Feb. 22 7 20 •••do.•' 103 8 9 Ill! 38' 33 :«HIll.... }lIfor. 211 30\11... 1 i 5 20 ...<10 •• ' 103 35 20 2~ 8~: 38 34 30.,3('.. Feh. 22 2' I 20 Dry••' \12 69 9 21 : 68 69 3033.... } I I 1 ! 303~ .... Feh. 2fi 0, 25 Wet..' 308 42 10 2~ 236 24 30 30.53B .. ~i , 3088.... }lIfllr. 20 308\L__ 2., 00.<10.. 20., 53 2~ 3;- 115 ' 23 ! 24 3030.... Feb. 8 25 Dry._: 59 , 52 52 1 j I oj 32 ------ '~~'I ~ I i . ---'-----'--: 1 (\) indicates small germs, (2) medium·sized germs, and (3) late morulae or early omhryos. For more exact do.,crlption of these stllges see Seasonal History, p. 49. The figures in the l'olumns under this heading in "The hosts, however, do not come Ollt of their winter lethnrgy ~C) I'I'ndlly III the fnll 0" I'lll'ly willtl'r ns the pnrll~itl·~. MACROCENTRUS GIFUENSIS, A POLYEMrlRYONIC PARASITE 55 certain that the parasites may emerge from the host, spin their co coons, and transform to adults under comparatively dry conditions. In fact, cocoons spun without contact moisture gave in general better emergence that those spun under wet surroundings. On the other hand. the adults seem to live longer in a moist at· mosphere than in a dry one (see p. 53). MORTALITY IN THE PREPUPAL AND PUPAL STAGES Under the method of rearing already described, the mortality in the cocoon stage under normal conditions was comparatively low. In a total of 297 colonies observed for this purpose, 200 colonies had no dead, while 97 colonies had one or more individuals dead in the late larval, prepupal, pupal, or adult stage. Adults to the number of 6,480 issued in good condition, while 691 individuals (or but slightly over 2 per eolony) were found dead in the 97 colonies of cocoons. These rea rings were made at temperatures of 18°, 20°, and 25° C., and the results approximate those obtained by field observations (see Limiting Factors, p. 57). COLD STORAGE OF OVERWINTERING HOST LARVAE CONTAINING PARASITES l'his experiment was undertaken in order to ascertain the possible effect of low temperature on the earlier stages of Macrocentrus. It applies to the late stages of the segmented egg,19 i. e., secondary germs and early embryos, and was conducted in the following manner: Lllrvue were collected in the field weekly, in lots of about 200, from February 1 to March 29, and placed in corrugated paper in metal shipping cans. They were immediately soaked by plunging the entire can in water. drained thoroughly, and placed in a constant temperature refrigerator at 4° C. Once during the experiment the cans were removed from the refrigerator, soaked thoroughly by dip ping, drained for 24 hours, and replaced. On June 24 all were taken from the refri~erator and, after removal of the dead larvae~ were soaked, drained for 24 hours, and placed at 25° C. When examined on July 9, a total of 130 colonies had issued from the host larvae und were spinning or had spun, while 136 colonies (as shown by dissection) in the second and third stages and one colony in the first stage were feeding int~rnally. Those consignments exposed to a temperature of 4° C. for nearly five months did not differ materially, in the percentage~ of parasites and chrysalids eventually produced, from those lots whICh were kept at 4° C. for about three months. The following figures summarize the data in this experiment: I Number Per cent I Per cent ------1------1----1 Larvae of P. nubilnlis in experiment. .•. _..•...._..•..•._._ •.•....•..•.••• 1,443 100 I...... Dead at end of cold'storllge lJeriod ...... _•..•._..•...... •..•...... •• 102 7.1 } 27 Died during illtter part of experiment•.....•...... •.•.•.....•....•.....• 288 19.9 Pupated.•.•..•••.....•••..••...... •...... •.....•..•.... 655 45.4 } Furnishing healthy p!lrasite colonies...... 268 18.6 i3 Henlthy Inrvae (unpurnsiti,ed) dissected lit end of experimenL ...... 130 9.0 ,. }I'or further data on the stages of development durIng this period see Seasonal History, p. 49. 56 TEORXICAL BCLLETIN ~30, IT. S. DBPT. OF AGRICULTOHE 'rIms it will be seen from this exFt~riment that cold storage at 4 0 C. cun be tolerated by both parasite and host, when supplied with moisture at intervuls to prevent desiccation, for a period of three to five months, Ilnd it will probably be found that they can tolerate sHeh stol'llg"e conditions for It much longer period.~o The impo"b\Ilce of the above data in the handling and coloniza tion of ill. giluensis is evident, when the manipulation of the para Hite and synchronization of liberations with desIrable field conditions is considered. COLD STORAGE OF COCOONS Data. relating to cocoons kept in cold storage are presented lJ1 Table 8. l'AllT.E S.-nal(/, on co('oons of Mu('rocent/"lls [/if1/.(,II.~is kept ill cO/ll .~forafJC tor di{f('fC'/Lt lJ('riO(l.y ·in 1!1£9 I Xllm· ESI.i.! I Tempern. Numher or hor or lIlllled, ]),lle IT Total ture tit ndults issuing colo. nllm'IPIIIC'Col in ('onolilionutstilrtorexpori. !,e,~;~ Date time in which ex· ____. nics in her of {'old stor· manL I t~;;e rcmo\"e51 ('old posod ",!,eri· indio ngo ' stornge subse· l\f I Fe· JllCtlt, vidunisr t quentl~-' • n es mules Dav. o C_ II: i 0 ~ ,/--'14: .\1"'. 2~" ~~o~u~",e lind "nrlrlm!'!!e'l C\ .Julle 1 40 IS 0 J20 1\11Il' J5! Lnte Inrvne colllpletel~' ·1 "rH~' :lI lfi 25 20 ISO 110._. i 1'.:'J;:~;;lleundeurIYPllPlle.! 4 .•.do .... lfi 2.j' H 'Z! (i0 \ :'-Iny ;- _ •• do •._.•___ ••. ~_ ••.••. I 4 .••clo •• 24 25 0 2 HX1,. do ... I I.lIlolllrvlIel'i1splln ...... j -I __ .clo .... 24 25 0 0 I~O f Ap~. 3()! rr~llUPIl,.e"n(.llllIrlrIJUJ1~··1 ~ '·J'u·',!,'c'--.';· a1 25 0 0 IhO 1 ...\I)I~ S!, plllmng lanne_._____ ~ .. ~~ "t liO 25 10 10 ISO I' ..do ••• ! COlll\,leteIY sJlun !olrme 4 00 1 fiO 25 '0 ! anI I'ropupae.• _"...... I ,...-- ' 0 I Wnnne" up slnwly. , Femules hud o\'illositors curled nnd were generally in poor condition, J l\'lullY dead were I1ott!u OIl June i, when removed from cold storngc. Thus it is e,-ident from these experiments that rhe late larval and pupal stages of this species ('un not well tolerate exposure to low temperature. e,-en :for so short a period as IG days. This method can not therefol"l' ue utilized in the manipulation and colonization of this parasite. MACROCENTRUS GIFUENSIS AS A CONTROLLING FACTO~ OF PYRAUSTA NUBILALIS This parasite appeal's to be of considerable importance as a con trolling factor in the l'imited areas in which it attacks P. nubilalis. Table V shows the extent of parasitism by this speCIes. ~~ Other material plnce,1 in cold "tornge nt the snme time wns furnishing normal colonies in abundance on Oct. 2ti, In!!!), when these pages were being written. ~UCROCENTRL'S GIFUENSIS, A POL1'EMBRYONIC PARASITE 57 'l'.IIILE O,-Perccllfu!lCs of parasitism, of Pyrau,sta nuliilnlis by ]fllcrocc'lItrlls !lifllellsi,~in 1'uriOllS ;:Ol/e,~ 1\l24 , , •• Ill!)'" • __ , •• ". ___ , 19:.!tl •• ,,_ ,,,...._ " __ ,, 1~.~17 ...... _.. _ .. ~ .",,, __ ~,,. ___ ~.",, . 1\12S ____._, __ ,'"______", 192!l______•••• _. ___ '_' ______1 I.\(hlltlonlll dntll ohtaill(',1 (rom dissectiolls lind rcnrings sincu the publication o( ~arlicr dlltll on this suhJcct (t9), LIMITING FACTORS The principal itl'm which limits the efficiency of .11. gifuensis as a controllin/! ia('ior of P. 1H1.vilalis is, in the writer's opinion, the inubilit~T of the female to locate and sting the host larvn. The reac tions of tIw f(,lIl1ti{' ill this ('onnection have be('n described under Oviposition. page 25, TIl(' writer Yentures to suy that in aU proba· bility nine-tenth,s of the eggs which the female is capable of producing are ne,-C1' deposited. . Mutual deSll'Uction among the first-stage larnle and the destrnc i ion of some large germs and embryos reduce somewhat the size of the colonies den'loping in a single host. It must be pointed out, however, that. as this destruction is greater when the host larva bears muny more parnsites than it can nourish and negligible when the size of the colony is below the average, this factor is not very significant. Mortnlity in the co('oon stage is not exceptionally high. In 35 • ('olonies collected in the fidd. 17 had no dend pupae or adults while t 18 colonies had 50 dead individuals. Experimental results (see p. :'5) with 29i colonies closely approximate these figures. Xo hvperparusites haye been observed in colonies collected from the field. RECOMMENDATIONS It appears to the writer that the methods of rearing and coloniza tion which would allow t.he optimum opportunity for establishment and dispersion of the parasite would be a system which envisages the following two principal points: (1) The liberation of material from I-generation areas (Europe) in I-generation areas. while the 2-gen eration areas would be supplied from material originating in 2 generation areas. (2) The most profuse liberations of the parasite ~ should coincide with or precede but slightly the maximum abundance of second nncl third stage host larvae of the owrwintel'ing generation. In order better to accomplish the latter recommendation, it will be found practicable to retard the spring development and emergence of imported mnterinl by cold storage. The acceleration of develop 58 TECHNICAL BlTLLETIN 2:10, U. S. DEPT. OF AGRICrLTURE ment can be started at a suitable period in adnmce of the appearallce of young host larvae.21 In this way the parasite would have a better chance to establish itself in numbers in larvae which overwinter. whereas. if liberated t'oinei(\ellt with the appearance of larvae of the' first ~el1{iration, many (·g:,..rs, owing to their slow rate of de,-elopment. would be lost through the midsummer pupation of these larval'. Thus the number of pal'asih's Ukl'ly to PIISS the first winter succl'ssfully would depend upon till' small proportion of host larvae of till' first ~enel'lltion which fuiled to pupate in SUmnll'I', and upon the number of ill, yifuenJ:tls, if any, able to de,-elop aml issue befol'e their first-generntion hosts had pupated,22 It is not considered advisable to retard the importation and. colon izution of ill, rfiluell~~is and other parasites in order to mnke ecological studies, On the contrary, no time should be lost in importing this parasite and libel'llting it at all strutegic points which present con sidl'rable easily determined l'Ilriations in physi('al and clnllatic char acteristics. provided that such lJoints are well supplied with accept able hosts, It is inad,·isable to libe1'llte the parnsite ill adnmce of till' borer spread, SUMMARY illucl'ocentl'llS gil/le-II,~is Ashm. is a primal'Y parasite of Pyrausta 1I1lbilalis Hiibn_ in Fram'e, Russia. and ,Japan, It hilS been reared from this host taken in Artemisia, corn, and hemp, It does not occm' as a parasite of P. nubilalis in the principal ('orll regions of Europe_ For this reason its economic importance ~l'l'mS at first glance negligible. The percentage of parasitism in the weed (Artemisia) areas of the Armorican zone of France has, howevl'r, lIlaintllilll'd itself around 30 per cent for several years; it is thus seen that this parasite is a factor which can not be neglected in the fight against the (,Ol'll borel'. The e:,..rgs of the parasite are laid in the body cavity of young host larme during the latter part of June and in early JUly, De \-eIOpllll'nt is by polyembryony. a method of deYelopment not before r('('orded in the Brnconidae, 'I'll(' egg. in till' writer~s opinion. tram;:forms to a tiny !-lpherical pregerlll. containing a huge paranuclear cell forme(! from the polar bodies and a small group (generally two to six) of embryonic cells derin'd from the ell'll\'agl' nucleus and contained within the ooplasm of the egg and polar bodies, i. eo. the trophamnios. The whole is encircled by a thick chorion, The pre~erm is usually lodged in a fnt cell. The pregerm ~..'TOWS to It primlu'y germ by the mitotic division IIf the embryonic eells, and this primary germ divides by fission. The paranuclear cell divides also. half of it accompanying each half of the germinnti,'e center, thus forming two secondary germs, (>ach of which i!-l similar to the primary germ, ,. ~'rom the moment of rl'lIlO\·,,1 from cold ~torage to the appearance of the adults, 3-1 d"y~ :It :!(i" C. nn' rl'tlulred. 54 dllYs nt :!O", und 60 days at IS", provided the material Is gin:~n Ihorolll.!'h :;onklu'!t; ac.·(,·ompllllhld h)r ult•.'rnnttl dryiJl~:-:;.· '" D. W. ,rOnt'~ hn~ r('nrl'.1 this purns!te from egg to adult. The minimum time re '1ulr('(\ for den'IClllnlC'nt fmm the l'gg to full-grown IlIr\'"al stnge was 14 days. This fact Is C'lh'd ll(ln~ in orllt'r to Ntr(lS~ tlH.1 h)·pOtiWHiH that ~t\ldit.'H m:l~le 011 an in~{'et in its IIl1th'" homl' mn lI<)t be tnk...n ns lin nhsolute guide to what thnt In~('ct will (\0 ill !L new en\·\t·oltment. 'rhls Is true for th., Ilnrnslte as -well as for the host. :'IACROCENTllUS GlFUENSISJ A POLYEMBHYONIC PARASITJo~ 59 The pamnudeus of the secondary germ now divides several times until its ('dis l'1ll'irdl' completely the germinative centel' of embl'yonic eells. The ~'mbryoni(' ('(>lIs continue their multiplication and the germi llutive center again divides to form two tertiary germs, both indosed within the h·ophamnio:-.. This pl'Ocess Illay be repented sevel'lll timl's and the resulting ger minative eenter:; !Wly rl'lIlnin together or separate entil'ely or in part. thus fOrm;{lg isolut(,d 1I1orulae 1 or parasite bodies containing from two to six Illoruhll'. l~s(,lHlogerllls or tl'ophumniotic mOI'sels, similar to the true germs excl'pt that t!l('y have 110 ('mbl'yollic cells, are present and grow lind divide to form hundreds of smaller ones. These pseudogerms do not develop into larnH~ but are eaten lateL' by the parusites. There is no blastula stnge. The morula changes into an oval disk with reflexetl borders and this elm'elops into a slender, euded embryo which fonm: the la rvu. TIl(', trophalI1nios degenerates into nn extremely thin membran" with flattelled Iludei. There is no I'l'oliferution of host tissue to fOl'111 a cyst as in 1~'II('yrtll$ jll,w'h'ol!i8 Dalm. The larvae glluw t.heir WHy out Hnd in so doillg they destmy some other IUI'vae all<1 gerllls. Hat<:hin/! tukl's pillel' in April. The first-stage larva has a head ami l;{ sl'gmellts. a pail' of sharp mandibles, four rows of enormous lob('s l'esl'lUbling pseudopoc\:';. und all evaginated rectum. The pe culiaritil's of it!' internal anatomy are the unequal lengths of the two Malpi~hia n tllbl'S and the distinct division of the hind-intestine into ileo-col(Jn and reetll111. Xo spirncles are present, though a tral'lwu! SystplU is den'lopecl. Tlw fin;t-sta/!l' lal·\'IH.' feed mostly on fat globules. The second and third stug(' lalTal' fepc! internally but the fourth stage issues from the host al\(I fel,ds l'xtel'llally. Thl' second-sfa/!l' larnl has no mandibles, the third has thin. weak ones, but the fourth stagl' has strong, toothed mandibles. Xo spir acles or tl'gunwlltary spines are present on the first and second stages but thl' f01l1'th-sta/!l' larva is covered with tiny spines and has nine pairs of open spiraeles. ,Yithin the ho~t, the larnle lie with their heads towanls that of the host. As thl'y is~·;ue. however. they reverse their position amI accom plish externnl f('eding orientated in It cauclal direction. 'While spin nin~, the position is again reversed so that the pupa is orientated cephalad. Thesl' ol'ientutions are the natural result of a series of m('ehanieal gyratiolls accomplishl'd in a certain manner. Pupation takes place about. the middle of .Tune and adult emergence towards the latter part of .June. The ovipositin/! femnle !!enerail'y lays one egg at one thrust, though sometimes perhap" two or three may thus be laid. Most parasitized l:uTae contain se\'ernl eggs because of the" re peating" habits of the femall's when o\·ipositing. As a result there :l1'e usually mally more Inrnle and w'rms of the parasite present in the host InlTn than it {'an nourish. The supernllmernrv germs or {anal' are de!'troyed during feeding and growth, " 60 'l'ECliNICAL nl'LLETlX 230, IT. S. DEPT. OF AGRICULTURE In these studies the aver/we size of a colony "'as 24 if all males, 16 if all females, and 20.9 ifs('xes were mixed; 37.5 per cent of the ("olonies were mixed bl·ootls. The maximulll number of indi"idual's dewloping- from It sing-Ie ('g-g is believed to be 8 or 10. The mortulity in the lnborntory rellrings of field-collected host mat('rial was 23.(j per cent, exclusive of those killed by parasites. The, Ityerage leng-th of life of females was 17.6 days unel the maxi mum "'IIi; 36 days when supplied with sweetelH'd or honey wat('r fot' fond. . Contact 1l10istlll'e fOt" tIl(' host in the spring' is nec('ssary for the I.h·,·elopment of the pnrasite, otherwise it will' die or produce ",mall, unlwnlthy colonies. TIll' tim(' reqllired to eomplete de"elopment from enrl,· spring' to 0 till' IIdult stng-e is 3+ days at :u;o c.. 5,1; days at 20 ('., lind GO day" at l/'i" C. TIl('st' tl'lI1pl'l'atures should be accompanied by alternate so.'lk ing-s and dryings of the host material. PIIl'I1sites ill overwintering host InlTne mny be retarded In' cold "torng-e for as milch as fOllr months or more after their normai eme'r /!el1ee time. Cold stOl,'a/!(' will kill the late lar\':le and pl1pae: however. LITERATURE CITED (1) AiHllIEAD, "'. H. loon. DESCllwrWNS OF ~a;w HYl{&~Ol'TERA FnOM .TAI'A:X. L. S. Xutl. lIu:,. I'roc. ao: 10!)....201. illus. (~) ('AFFla;" U ..1., lind \\'OItTllU:Y, L. iI. 11)27.•\ l'IU'G1IESS lt~a'llIl'l' ON TilE IN\,ESTIGATWN8 OF TIlE EnIO!'EA:" con:" nORER. U. H. Dept. Agr. Bul. 1476, 154 p., illus. ( :1) t'LSIDIAX, R. A. 11.11:1. BIOLOGICAl. XO'rES 0:" A n:w ltAltE 011 1.lrrUl KNOWX IlYlIENOPTh"R.\. ~ Ent. Soc. "'uRh. l'roc. 15: Hi3-1GO, illus. (-l ) FEilltli':nE, C. lO:!li. :xo'm 8l'1t 1'N CIfAr.CIIJl~;'" A uf:n;f.OI'I'E~U;NT POLYE~[[mYON[Qu~;. Rey. Ruisse Zoo!. a3: 585-5U6, illus. (;)) FINK. D. E. Ifl2U. Tin] lIIlll.OGY (W lr.\CItOC~;N1·Itt·S A:XCYL\,ORA ItO 11 WEll, A:X B{POIIT.\NT 1'.\IlASlTE O~' TIlt] STIL\WIlERRY LEAF ItOl.f.l-.,t (ANCYLIS COML'TA:XA ~·RlIEIIL.). Jour. Agr. UCst'areh 32: 1121-1134, illus. (til (;~:Nn;ys. 1'. H):!;). IIAIIIWlIItA('ON IIlt~)I'lCORNlS WESlI. Ann. Ent. Soc. Amer. 18: 14:} :W:!. ilIlls. (7) lI~:Iu:rf.A. B. ItJ2n. INSECT I'ARASITf:S OF TH~; ('OlIN BOREll IN NOItTHI-.,tX .Jt:GOSLA\'IA• I"tl'I·llIltl. COl'" Borer Inl'l·St. 2: 111-127. • (~) •T(lNE8. D. "'. U12n. DII'()ItT~:I) l'\lt.\SI'n;;; O~' 1'1I~; t:r'nol'~:.\:" CORN ROlUm E, .UIEItICA. U. S. Dl'pt. Agr. Teeh. Bul. 08, 28 p., ilIu~. IH) K(lItNIIAt:SElt. S. ,I. 1HIt). THE ;;~;XTAf. C}URAC'n:IlISTICS OF TIn] ~lElm\t.\CID, THELIA IJDUCu' LATA (~·ADlI.). I. EXTElt"Af. CIIAXG~lS INDUCED HY AI'HELOI'L'S 'rm:r.I.\E (GAHAN). JOlll'. .\[orph. 32: ;;:n-G3G, mil;;. (10) LI-.'"IIIY. H. W. 1!l2~. 'rHE l'Of.Y1DlIIIIY(J:Xrc D~:n:Lop~n:NT O~· coprooso~u GELF.CHIAE, WITn • Non:s ON IT;; mOLOGY. ;10111'. Morph. 37: 105--285, iIllls. t (ll) l!J::!G. 'rUE OIUGlN OF llIXEI) nROODS IN POf.YE~UlnYO"IC IIYlIEXOI'TrnA. ,Anll. Ent. Roe. Amer. 10: 200-2UO. (12) --- /llld lIn.f., C. ('. I!12::. THE TWINNINC, AND lroX~}~[BI:YO,,[(, DEVELOPllENT m' PLATYGASTER 1I1E~IALIS, A I'AltASlTE OF 'fUE HESSIAN FLY. Jour. Agr. Research 25: a:.:7~H!). iJlus. (];l) --- :lIId lIn.f•. C. C. 192-1. TIlE POLYE~rnltYONIC nl-.....·Er.OI'~IE"T OF PLATYGA.STER YEIt:XALIS. Jour. Agr. Ut'seareh 28: 82!)""s:30, il1us. (H) JXXfIIE. A. E. Jt)2-1.•-\ BIOr.OGICAf. STU)Y OF AI'IIELIXUSlf.H.r IIALD., ,\ PAItASITE OF THE WOOLLY APPLE AI'rnD. ElUOSOllA r.A:XlGrnA IIAUS~r. X. Y. Cornell Agr; Expt. 8tn. Mem. 7U, 27 p., illus. (15) '\!.\UCHAL, P. ] fl!)-1. IIECHEIICHE8 snt T.A lIroLOGTE E-r u: nf:n:r.OI'I'Elm"T fII,S rI\:~ff:"oP TI';;II~:S l'.\II.\SITES. I. r.A 1'0I~Y~;~lBltY()NH; SI'~:C[FIQeE OF GEn~n· NOGO"IF.. Arch. Zool. Expt. et Gen. CIY) 2: 257-335, illus. (H, 1 XELSON••T. A. In2-1. lIOItPHOtOGY OF THE IlONEYIlEE r,AI!\',\. Jour. Agr. ResenTl:h 28: 1167-1213. i1lu~. ( 17 1 f',HLr.OT. A. It)2<:;. ON TilE :XATUR.\£' EQUILIBnIU~[ OF l'YRAUSTA :XUIlIL.\US lIll. IlIt('rnat. COI'II ROl'pr JII\'est. Rd. Rpt. 1: 77-J06, illu;:. 61 l 62 'rECl[NICAI~ nULU:TIN :lilO, u. s. Dln'T. Ot' ",GHlCUL1TJ:E (1~) PAMI'!!:I., "', 11114, IIUl \\'l!:IlIl.ICIlEN' m:HCllU:OIlTSOKO.\NE IIEII H,'Il:n:t:lW:"lIIt::". Ztschl'. Wi,;;.;. Zool. lOS: [:loot-ilu7, illus. (ltl) PAIIKEII, H. I~. III:!". IUOC'IIEII('lIt:S SCIt I.t:s nlltMES l'OST-E~IIII1YU:":"_\lIIt::; liES t·IIAI.UIIIE:"S, Anll. Hoc. I~IIt. E'l'IlIH'C 1l:J: :!(lI-a!):!, illm:. (20) l'NI''''':lIIiON, ;1. 'J'. ll)Hi, Ollst;)"'Nrw:"s ON Tilt, I.E\'EI.OI'ME:"T OF COI'IIHI"t)~l.\ U~:U:t'lIl.\E. BioI. Bill. :lH ((i i : a::a-;{7fi, illlls. (21 ) 1!l21. SE.,{ ""TIOS I:" l'I..\l'YOAsn:lt. Aull'l'. Xul. ;:;;;: Isl}-l "'::. (2:!) HAlmA, It HUu. OSHt:ItVAZIO:"1 IIlOl. ORGANIZATION OF THE UNITED STATES DEPARTMENT OF AGRICULTURE WHEN THIS PVBLlCATION WAS LAST PRINTED 8e<;r('/II/'/1 ot ~L!1rl('lIltlln' __ . ARTHl'lt ~l. HYUE. ~lsxil Thi:o: hulll'till it; II cOlllriblltion from BI//,(,IIII, of JoJ/lIIIIIIQIO!I/I______('. L. lIAHLATT, ('lti"f. DI,I·i.~i')1I 0; ('/'/'('111,//111 For//lle I/I.~I'I·II,_ .. ,,,. H. LAHRI:.U:H, Prillcilitll EII IOllloloyl.• I, ill Glta,rye. 63 U 5 :iOV£RNMENT PRINTING OFFICE, l!fll .'