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TECHNICAL BULLETIN No. 230 ~ MARCH, 1931

UNITED STATES DEPARTMENT OF AGRICULTURE

WASHINGTON, D. C.

MACROCENTRUS GIFUENSIS ASHMEAD, A POLYEMBRYONIC BRACONID PARASITE IN THE EUROPEAN CORN BORER

By H. L. PARKER

Bntom%(Jist, Dirillioll of Ccrcal IIntI ]t'OI'a(JB n/"~cct8, BII/'eal of Entolll'()/o(Jy 1

Disrowr)' in Enrol'" lind id,lttltr 01 th~ ~I)eli('.s _~ .. _",. _" ~ ~ ~ " ~ .. ,_," _.. _______ .. ______ ... ..

Om1b'faphic distribution !lltd host fllations .. . lIi;;tory 01 poIY~lllbr;'on)' in Hymenoptl'r!L .Iredu ic (ollowlld, __ , ... __ ~ ___ .. __ ____ .. _* __ 0,. __ ExpilLll!ltinn 01 symhols U~l'tl in thl' illu,tr!l'

tlOns................................. __ ... Descriptinn 01 .'[(lcroctlll",. ui.{ue/l~is .. _"'"

The !ldult............................. ..'l'he l~gg. __ ___ ~ __ ~~_ .. ____ ~ __ ~ .. __ . _.~_ .. _ 1"rhl'lnrvLL. __ .... ___ ......... ___ ._.o ___ ...... ... rl'hc pnlJ)upn unu pUpil. ...... ~ __ .. ~ .. " ... __ _ 'PI10 cocoon_____ .._.... _.... _.. __ ~. __ .... ~" .. * _. __

.Bit)lo~y.............__ ._..................... EtIu.\r~lnL't. __ .. ~ .... _ ...... __ ... __ ~_~_~ ... _ L,ght rmctions .... __ ................ . Feeding....... __ ............... __ .... . Oogenesis____ M _. ~_. .. .. _". ~ _~ 4 .... .._ _ , ~. _

~~\~~~~~\W~':i:.~:: ~:::::::::::::::::::::::: ~t:gml'ntation of tho (I~g _. " ................. ~ ~ Growth and dcv\lopull'rt.. of tlw l'~g ~_. 0" P~clldogl'nns~ .. _....... 0 ..... ___ .... ___ ______ _

Cyst lormlltion ......................... . Lllrml hnbits............................ ElTect 01 pnrnsitl' on ho.,t IIInn .... , ... .. Spill' ing .. nd (lUp' tion ..................

CONTENTS

Pag~

Biolog\'-Continued. A v'emge ~ize 01

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

3 l\IACROCENTRUS GIFUENSIS, A POLYEMBRYONIC PARASITE

nucleus, the CC!ljugation of the male and female pronuclei, the COIljugation 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 embryonic 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 parasitic Hymenoptera is presented in Tables 1 and 2.

'l'hi~ phn~e (If denJoplIll'llt will be mude the subjpct of further researches.

http:summa.ry

~

TABLE i.-Parasitic Hymenoptera /;nown to reproduce by polyembryony

--~r-- Xumber

! formed rOIll oneAuthority for polyem 'I'Pumsite spe~ies lIos! egg orsl~t~!~I,~~;,Yry i hryony in the spedes , issuing

I ,from one [ host ,----------------

('halcidoide:1.' .\gcniospis fuscicollis :\lllrehnl (I.;) Hyponomeutus mnli- ! 150 (Dulrn.) 'l'h. nellus et al.

Do. _ .\, fuscicollis praysin Silvestri (25). __ ., Prays oteellus Fnll. 10 coin Sih'estri. '

Do. , I.itomllstix trunlO\tel :;ih'estri (E.I) _ PlusingumrIl!l L l,liO() Ius Dnlm.

1)0. Ageniaspis testoceipes :\Iorcllul (15) ...... LithocolletL~ cramer. Ii 12-15 ]illtzb. I elln,

Do_ . ('opidosoma bnyssoni ' Sih'estri (6, Coleophoru stefonii j 41-11~ :\Inyr. Jonn. ,

1)0 ... ('opidosoma nanellne : Sih'ssUi (E8.). ' Recnn'arin nan ella I 3-11 :-iii\'. IIilbn.

1)0. _ .. _., ('opidosorna geleehill. Leiby (10), Patterson II G norinlOschemll gal I 165 lIo\\,. (:?O). Illmolidoginis Hil. I

L])0 _._. l'opidosuma boncht Parker (19)"_' __ "" __1 Oelechin pingninelln 38 anUIU Rlltz. '!'reits.

])0_ Copidosoma thomp ,...do..._...._ Nothrix senticetella j 18 soni 1\[erc. I Stgr.

DIl_ I.itomnstixkriechbnn.: Ferriere W ......." ... Depressaria alPigen'l 21-121) merIMllyr.' ella Frey.

Serphoidea. I'olygnotus minutu~ :\Iarcbal (15) .... _.... Phytophagu destruc , 10-12 Limlrn. tor Say. I

;;; t':I o IIInrief summary of details and peculiarities of development ~ ..... n:.C"

Polar bodies form paranuclei which (in trophamnios) encircle germ cells at or q t:;j

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.

Similar, except that paranuclei from polar bodies do not encircle germinati\'e cells ~ until 12-celled stage.

:legmenting egg differentiates into two distinct parts; one part from the polar c-:'" 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 polyruorulneand 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

Advanced stages found in cyst ofhost tissue similar to that caused by A. fusclcollis. t':I I-d

Probably similar to A. fuscicollis. I'aranuclei do not encircle germinative cells !"l before 14-celled stage.

Embryonic chains and asexuallar\'ac observed by Silvestri. o >tj Polar bodies form paranuclei. Blastomeres become grouped in groups of about ::

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

Develops in \'isceral cavity in chains resembling those of A. fusricollis. Asemnl larvae pr~ent. ~ ;;;Similar to C. boucheanum. ::\[",Ie unknown. q

:::l t':IChains of developing embry~s observed in hody ca\'ity.

In morulustage the peripheral cells become detached to form the paranuclei of tbe 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:; 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. 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-formingII

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~ter hiemali~ ! 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

:::l, I daughter germ. The 2 daughter germs separate and give rise to 2 embryos. oDe\'elopment takes plat'e in visceral cavity. 'l'bis species may also develop byI, nmonembryony and is considered an example of the most primili\-e method of t:jpolyem bryonic development.

\'ariahilis Leihy (I1)- --... ' . ~i Hhopalomyill mrolin;l. 15 Early stages not elucidated. Z DO"-"' i PI~~J:ter H

I ::::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). rr.Paranucleus divides and balf accompanies each germ. Tbe paranuclear cells multiply by amitosis and yia the tropbamnios they e\'entually encircle com opletely the germinative center. Tbe secondary germs again divide to form tertiary germs or morulae, wbleb by multiplication of embryonic cells transform ~ q 2 l

Z {fl ..... .5"TABLE 2.-HY71lcnopiera Sli'~1)ecied of polyembryonic reproduction

'"j.' I .-"l~l1l,!brr ;,

of am,ly or I I'"ro.,te Sl)~",n, ',,'I,()r',l\' 11

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 experiments 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

7 l\:t:AOHOCENTRUS GIFUENSIS, A POLYEMBHYONIC PARASITE

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 histologicnl 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

8 TECHNICAL nrLLETIX ~30, U. S. DEPT. OF AGHICFLTURE

sp, sensory pllpilllle. 11'0. trOJlhllmllios. .~t, stomnch. II, urate cclls . f, trnns\erse. 1/('. unstained edge. th, thornx. 'l\ Y:tcuole. fill, trnusverse muscles. '!'I/o. yentrodorsnl obliquc. flllt, transverse muscle fibers. 'l:i, ventrolateraL to, tentorium. '1:11, yentroluteral lobes. fr, tntchcll. 1t, ventral. tnn, trophnll1nios dcgcnerated into a I 'Wilt;, Willi of mid-intestine.

thin llwmbl'llnc.

DESCRIPTION OF MACROCENTRUS GIFUENSIS THE ADULT

The adult (fig. 1) is described by Ashmead (1, p. 191) as follows: J.'clllalc.-I.en~,'th, 4.5 mm.; OYllloitor longer than the whole insect. Head

bla('k; t.horax brownish rdlow, the metathorax du,:ky or brownish; ubdomen

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, brownish yellow, becoming dusky toward apex, 40 or more jointed, the joints dusky

9 MACROCENTRI:S GIFUENSIS, A POLYEl\IBRYONIC PARASITE

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 blackish 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

FIO{JIlE :!.-Eggs of(the end directed dowllwal'(ls in the o \'11ry ) ,![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 }'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 segnwnts, and antel'iorly the Ill'shy mouth parts protrude as if "over

"Fink (5) IUl6 til'scrll>ctl the tlrststn~" Inrvll or M. a/lel/lieora. His 1'igllre -l r('sembkRbut little his (lhoto~ruphs (tl~. :~. U) of the flrststn~e Innn. 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 papillae; the labium (fig. :3, C, lb), which has two similar protrusions

\I

JI

lo'l

1\IACnOCENTHPS GlFUENSIS, A POLYE~fBRYONIC PARASITE 11

The body propel' i!-; upproximately cylindrical, but the presence on eneh of Segments I to XIII of four enormous fleshy lobes gives it It l'Iltlwr fllltteneli nppelll'ance bearing a superficial resemblance to t'ertain lsopods. (Fig. 3, A.) These lobes are placed along the dorsolaterlll Ilnt! ventrolateral lines, und all pail's extend in ventroIutel'lll und dorsolate1'll1 dileeti.ons, except the yentrolaternl pair on

...,'

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; Segment, 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.-Secondstnge l:um In outline, sliowing digestive tract. The ~all\"llry glands and ~Ialplghinn tubes arc IIbbrevlated. B.-Thlrdstage larvlI,side view. C.-!.'ourthstage 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 Segment 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 ventrul floor for It shut't distance, rises somewhat, and again brunches into two prineipnl trunks in Seg-ment IV. Each of these trunks subsequently mmifies in all irregulllr manner into dorsal branches of nlrying length.

The tl'llchenl system (fig. 4, D) is composed of two latera,llongitutlinul trunks cOllnected anterior! V by 11 dorsal transverse commissnre located in the first segment 1I1Hl 'posteriorly by It ventral commissure 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 extending 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 2lobecl 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 representee 1by two small ()'ml gonads locuted in the posterior part of the vis('('1'Hl cavity,

-----.-~.---------------

"J'h.ls Ilrutl't1~IOII

14 TECHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE

Te!!umentary muscles ca.n be observed extendinO" from se(~ment to ~ ~ 0 0

seg.ment, but ,none are present in the lateral lobes. They are dest'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, interspersed 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 mandible 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. Theirreaularity 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 i1eocolon-is cvaglnllted.

http:de~cripti.on

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 dorsally 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 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 l3Uriaces, 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 spiraell.'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, Segments 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 specimens. 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 cutide. It is ullifonn in thickness save ill the head, where it is considerably 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 reference 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 conspicuous 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 appendages (fig. 7, D, ill,.), located ventrally in Segments XI and XII in the female larva, and in Segment XII in the male larva. The imaginal 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

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18 TEOHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE

hypodermal imaginal cells are noticed in abundance in- late fourthstage 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!sS: 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 section through posterior end of hody "f Jur\'a slightly to left of medhln line; E, grossomodo view of mnle Ilnd .female rC'pro:luctlve organs In the larva; F, section throughgonad 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 commissure to supply the rectum, muscles, and the other organs and cells of the segment.

st

spr

esp

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 throughsulivury 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 connecting 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 reproductivenpPI'I)(Hlgcs which IIrc suppllM with nerYl' branches from the termill,,1 gnngllQn.

14

http:yolullliuc.us

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 esophagus 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

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 ileocolon 01' anteriol' pOltion and the r~ctum or hindcL' PIll't. The ileocolon, 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 sal i\'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\'(\rtxt('!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 posteriorly and somewhat laterally to the lower wall of the pharynx. One pair of ruther fruil muscles extends from the tentorium posteriol.'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 ventrally 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

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 segment 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 extending longitudinally between the stomach and the layer of tegumentary musdes as well as small pieces between the hypodermis nIHl muscular 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 mklintestine. Th('t,C' are no urate cells ill the thontx. ThC' oenocytes (lig, 6, A, 7, D, 0(,) nre found singly OJ' ill groups, principally laterally HettI.' the spiracles.

THE PREPUPA AND PUPA

FIGURE D,-Three cocoon colonies of .JlI/creaentr"8The prepupa, and pllpa {JifuCIISi8. '.rhe sbriveled skin of the host may be

I, tl I 1 He,'n nttached to the lower hRlt of ench of the are 0; 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 gregariously 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, however. 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 different 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 maneuvers; 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 sometimes 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 thatOllghly 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 oviposition 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 oviposition 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 ovipositor. 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 somewhat 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 ovipositor in its tip at a point about one-third to one-half of the distance from the base and seems to direct that organ in the forward and sidewise exploration thrusts. 'Whether or not the sheath actl1ally 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 present she becomes excited and her thrusts become more frequent; if the point of the ovipositor comes into contact with the larva it is thrust into the latter, an egg is quickly laid, and the ovipositor is almost immediately withdrawn, straightened out, and sheathed.

l!'loullm lO.-Fcmnle MaCI'occlltrus oi1"ensls in Because of the manner inposition 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 second 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. Occasionally 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 oviposit 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 preliminary 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 contortions of the body and by retreating farther into its burrow. If the burrow is shallow and the larva can not retreat, it leaves the burrow 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 larya 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 efforts 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 interior. 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 between the hypodermal cells and the cuticle. It is possible that some are placed in the digesti,-e tract, but these would probably be evacuuteu.

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 probable 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 exceptions, 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 generally 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 dissected 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 without 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 singleegg 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 following 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 central 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, represents 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 9'that these three are the polnr bodies which will later form the paranu

prndeus, while the foUl" larger ones are blasto1I1(,l'es.

A.nother unstained (i:\('gg observed ill physiological solution is illus ~ ode.ttated in Figure 11. B.

l"IGl:ltE 11.-1'gl;s of MaCI'OCCllirIlS girucII8is in differentaIld the following' de~ stnges of dC\'clopm~nt: A. Egg le~s than 5 hours old s("l'iption applies to it: (BoniJl's); B, egg Jess tlllln 5 hours old. showing

complete segmentation; C.

30 TECHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE

to the smaller ones in the IS-celled egg. This egg has conglomerations 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 germinative 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 previous 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:} ('ran ular matl'ri aI, the trophamnios (tro) l ill which is embedded t! farn dissociates and the::e cells scatter in ,yroups of from one to four. When b\'o or more cells cling to(Tether, or come together after separation, thus forming a group, a pregerm is formed, provided there is present at least one cell of each 801"t; a polar edl (or cells) without a blastomere gives rise to a pseuclogerm. This hypothesis wonld explain (1) the small size of the pregerm as compared to the original egg, (2) the small number

MACROOENTRUS GIFUENSIS, A POLYEMBRYONIC PARASITE 31

of cells present in the pregerm, and (3) the presence of the pseudogerms.

The second hypothesis, which seems more probable, is that each pregerm represents an entire egg which has progressed to a certain

gm prn tro 9c tro

'gc

l1fc.

nfc B

~h ",~:*o---,,;.;;

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.

http:tuhryon.ic

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 rapidly than othel's and furnish pregerms snch as those shown in Fig

nfc

--", ...

(JIj,"

lro A'

l .;

" 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();.aI~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'prlllinnthc ('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

t

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 polyernbl'yonic ('halcid, and shows thnt the prcger'm is quite similar to that produced ill 111, flifuCl1sis, The same author (128) has illustTlited 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 keptlit 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 COllsillel'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 prel'mbl'Yo whieh the write I' hit!:; observed, and it establishes polyembryony. The two daughteL' germs arising from this division are hel'e ('ulled sc('olldary gl'1'1I1S. Figure 13, C, is an example fr01l1 fieldeollect:('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 germi nati \'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

34 TECHNICAL BULLETIN 230, U. S. DEPT. OF AGRICULTURE

phamnios, which is absorbing material from the host blood, increases somewhat in thickness. (Fig. 14, C.)

'When the germinative center of the secondary germs reaches that stu.g-o of growth in which it is composed of about 200 to 300 cells it agajn divides in two parts to form two similar germinative centers or tertiary germs contained in the same trophamnios. (Fig. 14, A.)

These germinative centers may de.ve.lop to embryos or may perhaps divide once or l:icveral times in the same manner, thus producin~ parasite bodies containing 2, 3, 4, 5, or 6 germinative centers snell as those represented in Figures 14, B, and 15, A.

.....~,.~ .... .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

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 reular 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 center 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 3morula 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 polymorulae.

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

36 TgCUNICAL BULLETIN 230, U, S, DEPT, OF AGIHL'LLTUHE

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~ becomes 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 trophamnios has

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 lilllorllta 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. ptlrltIIlP!l'US; jJ.l(glll, IhPlldoL;I'rttJ. try. (r lJ!lIh'ul. Itu, lrol'h'HuHio... ; I, \ 'wtlflllj ~

}\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 showIng 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 hatching time when large number's of small ones having fiye to seven paranuclei 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. testaceipes, 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 ~ ,"" " ''''' "",., e "" oody. Such is not the cuse with Jlf. gifuenS'is. There is no such prolifOl'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 coineides 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'(~ displnC'(\(1. Tlwir walls and. eontcnts thus form It sort of impI'ovised eyst of mesellchymatow; 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

-------------------------------

MAonOOENTRUS GIFUENSIS, A POLYEMBRYONIO PARASITE 39

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 extremity, 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 inhibited 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 destruction 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 N I1 um }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 2 __ a i- ----~ M_~~ ____ ______ _______ __ '.'.' :_":' _ 7 25 ,~ ........1~~ .. _____ _____ .. ~~ --------__ .-~--.--- --------___ _____ ~ ....

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 germinativecells 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 permanentIyin 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 migrate 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 following mannel': 'rhe capsule splits, owing to the pressure from within, nnd the head of the fourth-stage larva is lifted out of it. 'rhis cnpsuit', 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, however, 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,

http:globules.12

: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 dislurbed by emergii1g larvae. After this period of repose, the larva becomes restless and manifests this condition by dorsoventral movements 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 material 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. Microscopic 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 Malpighian 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 longitudinally 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 ventral 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 completely 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 connection that this sudden change i