\ Pergamon Int[ J[ Insect Morphol[ + Embryol[\ Vol[ 16\ No[ 3\ pp[ 214Ð220\ 0887Þ 0887 Elsevier Science Ltd[ All rights reserved

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CELLS RELEASED IN VITRO FROM THE EMBRYONIC YOLK SAC OF THE STICKINSECT CARAUSIUS MOROSUS "BR[# "PHASMATODEA ] HETERONEMIIDAE# MAY

INCLUDE EMBRYONIC HEMOCYTES

M[ Teresa Locci\�$ Massimo Masetti\% Antonella Cecchettini= and Franco Giorgi=

$ Department of Biomedicine\ University of Pisa\ % Department of Ethology\ Ecology\ Evolution\ University of Pisa\ = Department of HumanMorphology and Applied Biology\ University of Pisa\ Pisa\ Italy

"Received 15 March 0887 ^ accepted 09 Au`ust 0887#

Abstract*The embryonic yolk sac and the adult dorsal vessel of the stick insect Carausius morosus "Br[# "Phasmatodea ]Heteronemiidae# were shown to release a number of cells that appear morphologically similar to circulating adult hemocytes[Like adult hemocytes\ these cells reacted positively when tested for both phenoloxidase activity and a monoclonal antibodyspeci_cally raised against a vitellin polypeptide[ Based on this evidence\ it is suggested that yolk sac!released cells behave aspotential embryonic hemocytes[ A model is thus proposed whereby the yolk sac might host a number of hemopoietic stem cellson their way to the dorsal vessel\ and in so doing\ it may temporally act as an embryonic hemopoietic organ[ Þ 0887 ElsevierScience Ltd[ All rights reserved[

Index descriptors "in addition to those in the title#] embryo\ hemocytes\ hemopoietic tissue\ stick insects\ yolk sac[

INTRODUCTIONInsect hemocytes comprise several morphologicallydi}erent cell types playing a wide range of immunologicaland physiological functions "Gupta\ 0874\ 0880 ^ Go�tzand Boman\ 0874 ^ Ratcli}e et al[\ 0874 ^ Marmaras et al[\0885#[ Various histological and histochemical techniqueshave been employed so far to determine the nature andlocalization of hemopoietic tissues both in adult andembryos of insects "Feir\ 0875 ^ Mori\ 0868#[ The majorconsensus to date is that adult hemocytes originate froma number of hemopoietic isles associated with the dorsalvessel "Brehe�lin\ 0862 ^ Francžois\ 0864 ^ Ho}mann et al[\0868#[ In addition\ there are loosely organized hem!opoietic tissues\ as in Locusta mi`ratoria "Ho}mann\0869# or comprising only a few clusters of di}erentiatedcells circulating throughout the bloodstream\ as in Cal!liphora "Ho}mann et al[\ 0868#[

During early development\ embryos are overlaid by ahuge vitellin laden yolk sac that is gradually depleted ofits content as vitellin is progressively utilized by limitedproteolysis "Fausto et al[\ 0883#[ Because embryonic yolksacs of the stick insect C[ morosus have been shown torelease a number of di}erent cell types in culture "Locciet al[\ 0884#\ the aim of the present study was to seewhether some of these cells might behave as potentialembryonic hemocytes[ In order to do this\ the cells soreleased were examined morphologically and compared

� Author to whom correspondence should be addressed[ Departmentof Biomedicine\ Laboratory of Pathology\ University of Pisa\ ViaRoma\ 44 45015 Pisa\ Italy[ Tel[] ¦2849 490967^ Fax] 2849 490974^e!mail] mtlocÝbiomed[unipi[it

214

with adult hemocytes as well as with those released fromthe dorsal vessel[ In addition\ reactivities to phe!noloxidase and to a vitellin polypeptide antibody wereexamined both in yolk sac!released cells and adult hem!ocytes[

MATERIAL AND METHODSAnimals

Specimens of the parthenogenetic stick insect C[ morosus were main!tained in a rearing room at constant temperature "19>C# and fed onfresh ivy leaves[

Hemocyte collectionHemolymph from adults was collected by piercing the cerci and

diluted 0 ] 0 with Grace|s medium[ Hemocytes were pelleted by cen!trifugation at 149 g for 4 min and rinsed twice in phosphate bu}ersaline "PBS#[

In vitro culture of yolk sac and dorsal vesselYolk sacs were dissected from 8Ð00 week!old embryos of C[ morosus[

They were immediately transferred to pierced sterile glass slides\ cen!trally sealed with a thin coverslip\ and incubated in Grace|s mediumcontaining 099 mg:ml streptomycin and 099 U:ml penicillin[ They weremaintained at 14>C without CO1 for 17 days\ and checked periodicallyfor their structural integrity[ Yolk sacs and the cells spread over thecoverslip were subsequently collected and processed for SDS gel elec!trophoresis and electron microscopy[ Dorsal vessels were dissected fromadult females and cultured under the same conditions as above\ withcell!free adult hemolymph added to the medium[

Sodium dodecyl sulphate polyacrylamide `el electrophoresis "SDS!PAGE#

Samples were dissolved in 49 mM Tris!HCl bu}er at pH 5[7 con!taining 4) b!mercaptoethanol and 4) SDS\ boiled for 2 min andthen applied onto a 4Ð04) polyacrylamide gel gradient[ Acrylamidesolutions were prepared as speci_ed by Laemmli "0869#[

M[ T[ Locci et al[215

Fig[ 0[ "A# SEM micrograph of a yolk sac cultured for 01 days[ ×29[ "B# LM photograph of cells released by the yolk sac after 03 days of culture[×049[ "C\ D\ E# SEM micrographs of cells released by the yolk sac as pictured after 4 days culture[ C � ×249 ^ D � ×0499 ^ E � ×0999[ "F#

SEM micrograph of yolk sac!released cells spreading on the glass surface after 03 days of culture[ ×249[

Western blottin`Following SDS gel electrophoresis\ polyacrylamide gels were placed

onto nitrocellulose sheets and electrotransferred for 1 h at 05>C accord!ing to Towbin et al[ "0868#[ After electroblotting\ nitrocellulose sheetswere treated with monoclonal antibody Mab 01 as previously reported"Giorgi et al[\ 0882#[

Transmission electron microscopy "TEM#Yolk sacs were _xed in Karnovsky|s _xative for 1 h and post!_xed

for 1 h in 0) osmium tetroxide in 9[0 M cacodylate bu}er at pH 6[1[Samples were then dehydrated in a graded series of alcohols and soonembedded in Epon!Araldite[ Five hundred A� !thick sections were cuton a Reichert ultramicrotome\ collected with Formvar!coated coppergrids and observed in a 0199 Jeol EXII transmission electron micro!scope\ following conventional staining with uranyl acetate and leadcitrate[

Scannin` electron microscopy "SEM#Yolk sacs and cultured cells adhering onto the glass coverslip were

_xed as above and then critical!point dried in a Balzers| CPD 919apparatus equipped with a liquid CO1 inlet[ They were then mountedon aluminum stubs and metal!shadowed in a Sputtering Unit "Balzers#equipped with an Argon inlet[ Samples were eventually observed in a4199 Jeol JSM scanning electron microscope[

Phenoloxidase activity assayPhenoloxidase activity was assayed according to Huxham and Lackie

"0875#[ Hemocytes collected as above were placed on 05!well plates"Falcon CultureSlide\ Becton Dickinson Labware\ Franklin Lakes\ NJ#\allowed to settle for 59 min\ then rinsed and incubated for 0 h in 049 ml

Grace|s medium containing 0) laminarine[ Yolk sac!released cells werealso allowed to settle for 6 days in 05!well plates and subjected to thesame treatment[ Fifteen ml of 4 mg:ml L!DOPA were added to themixture and cells photographed after 3 h of in vitro culture at roomtemperature[

ImmunohistochemistryCells displayed on 05!well plates were _xed for 29 min in Karnovsky

and rinsed twice in PBS and allowed to react with Mab 01 for 59 min[Following a thorough rinse in PBS\ cells were treated with a peroxidase!labelled goat anti!mouse antibody[ Enzyme activity was revealedthrough dyazol staining in presence of hydrogen peroxidase andobserved under a Axiophot Zeiss light microscope equipped withdi}erential interference contrast objective lens[

RESULTSWhen cultured in Grace|s medium for 03 days\ the

yolk sac maintains its overall morphology along with thecapability to undergo rhythmical contractions "Fig[ 0 A#[Under these conditions\ a number of cells appear in theculture medium with a minimum time interval of 1 days"Fig[ 0 B#[ The cells so released range in size from 19 to29 mm and are either roundish "Fig[ 0 C#\ slightly spindle!shaped "Fig[ 0 D# or show numerous _lopodia over thecell periphery "Fig[ 0 E#[ This morphological het!

Embryonic Hemocytes of Carausius morosus 216

Fig[ 1[ "A# TEM micrograph of a freshly dissected yolk sac showing the presence of several cells containing electron!dense organelles externallyto the basal lamina[ ×3999[ "B# and "C# are SEM micrographs of the yolk sac wall showing 1 cell types overlying the muscular wall[ B � ×0099 ^C � ×0599[ "D# Enlargment of a yolk sac!released cell showing several electron!dense granules in the cytoplasm[ ×09\999 ^ "E# Hemocytes from

adult female hemolymph showing several electron!dense granules over the cytoplasm[ ×09\999[

erogeneity suggests that\ during culture\ the yolk sac!released cells may modify their overall shape and changethe extension of their peripheral cytoplasm[ After a per!iod of 03 days\ cells became so numerous that they formeda cellular monolayer "Fig[ 0 F#[

At the ultrastructural level\ the yolk sac is characterizedby the presence of numerous cells lying externally to thebasal lamina "Fig[ 1 AÐC#[ Some of these cells appear tocontain electron!dense cytoplasmic organelles "Fig[ 1 D#\which are similar in number and content to those presentin adult hemocytes cultured under similar conditions"Fig[ 1 E#[ Hemocytes freshly dissected from adult stickinsects may in fact be either globular with numerousgranules bulging from the surface "Fig[ 2 A#\ ~at androundish with several pseudopodia and _lopodia pro!truding all around the cell periphery "Fig[ 2 B# or even

elongated and spindle!shaped\ with one or both extremit!ies attached to the substratum "Fig[ 2 C#[ On the basis ofearlier studies in stick insects "Scapigliati et al[\ 0882#\these cells are likely to be granulocytes and plas!matocytes\ respectively[ Like adult hemocytes\ yolk sac!released cells are capable of adhering onto the substratumby protruding several pseudopodia along the cell periph!ery[

In stick insects\ the dorsal vessel is a straight tubularstructure\ which lies along the dorsal midline of the abdo!men "Fig[ 3 A#[ Hemopoietic isles in this structure com!prise several polymorphic cells that are embedded in aloose _brillar network surrounded by numerous peri!cardial cells "Figs[ 3 B!C#[ At about 3 days of culture\ thedorsal vessel starts releasing several hemocytes into theculture medium that attach readily to the substratum[ In

M[ T[ Locci et al[217

Fig[ 2[ SEM micrographs of adult hemocytes[A � ×3999 ^ B � ×0499 ^ C � ×0999[

the scanning electron microscope\ these cells appear aseither roundish or elongated "Fig[ 3 D#\ sometimes pro!truding with several pseudopodia along part of the cellperiphery "Fig[ 3 E#[ Like the cells released from yolksac\ hemocytes tend to cluster\ forming large cellularaggregates "Fig[ 3 F#[ Based on these observations\ weconclude that yolk sac!released cells share some mor!phological and behavioral characteristics with the hem!ocytes released from the dorsal vessel[

When adult hemocytes and yolk sac!released cells weretested for the presence of phenoloxidase activity\ bothcell types appeared to react positively by forming a darkreaction product over the entire cell surface "Figs[ 4 AÐB#\ suggesting that this enzyme activity may already beexpressed during early embryonic development[

Extracts of adult hemocytes and yolk sac!released cellswere resolved by SDS!PAGE and tested against a panelof monoclonal antibodies reacting with a full complementof vitellin polypeptides in stick insects "Giorgi et al[\0882#[ Under these conditions\ vitellin polypeptide A2

appears to be the only polypeptide detectable in both cell

types "Fig[ 5#[ Adult hemocytes and yolk sac!releasedcells were also tested immunohistochemically with Mab01[ Data show that both adult hemocytes "Fig[ 6 A#and some of the yolk sac!released cells "Fig[ 6 B# reactpositively with the antibody[ Based on these obser!vations\ we conclude that hemocytes and yolk sac!released cells share the same capacity to incorporate vit!ellin polypeptide A2[

DISCUSSIONThe present study has shown that in vitro cultured yolk

sacs and dorsal vessels from the stick insect C[ morosusare capable of releasing several cell types into the culturemedium[ When obvserved in the electron microscope\ thecells adhering to the substratum were found mor!phologically heterogenous\ comprising at least 1 di}erentcell types[ One\ which is globular\ appears to adhere tothe substratum through the entire cell periphery and tocontain several electron!dense granules[ The other is spin!dle!shaped with several pseudopodia at both cell poles[Thus\ when examined under similar in vitro culturingconditions\ yolk sac! and dorsal vessel!released cellsappear morphologically comparable to adult hemocytes"granulocytes and:or plasmatocytes# of C[ morosus[Adult hemocytes do in fact include globular cells\ suchas granulocytes and spherulocytes\ and spindle!shapedcells\ such as plasmatocytes "Scapigliati et al[\ 0882#[Moreover\ the possibility for adult hemocytes to adhereto the substratum and to protrude with several pseudo!podia and :or _lopodia along the cell periphery has beenpreviously reported and documented by light microscopyin several insect species "Mitsuhashi\ 0861 ^ Huxam andLackie\ 0877 ^ Pech et al[\ 0883 ^ Gupta\ 0886#[ Insummary\ from the data reported in this paper\ it is evi!dent that cells released by both the embryonic yolk sacand the adult dorsal vessel share a number of mor!phological and behavioral features with adult hemocytes[

To check whether these cells are not only mor!phologically but also functionally similar\ they wereexamined immunohistochemically using several cyto!logical markers[ Very few studies have so far dealt withembryonic hemocytes\ while adult hemocytes have beenimmunohistochemically and biochemically well!char!acterized "Gupta\ 0874 ^ Ratcli}e et al[\ 0874 ^ Marmaraset al[\ 0885 ^ Scapigliati et al[\ 0886#[ Phenoloxidase isan enzyme activity previously detected in hemocytes ofseveral insect species "Leonard et al[\ 0874 ^ Iwama andAshida\ 0875 ^ Marmaras et al[\ 0885#\ that acts in cell!mediated immunity to detoxify the hemocoel fromexogenous agents\ mainly bacteria[ The fact that hem!ocytes and yolk sac!released cells react positively for phe!noloxidase suggests that these latter cells may also beaccomplishing the same role in embryos[

Another interesting characteristic that can be exploitedto identify hemocytes is the capability to take up proteinsfrom the hemolymph "Marmaras and Tsakas\ 0877#[ Inadult insect females\ vitellogenins are transferred from

Embryonic Hemocytes of Carausius morosus 218

Fig[ 3[ "A# SEM micrograph of adult dorsal vessel[ ×14[ "B# SEM micrograph of hemopoietic tissue localized along the dorsal vessel "arrows#and comprised between the pericardial cells "arrowheads#[ ×0399[ "C# SEM micrograph of a di}erentiated cell of adult hemopoietic tissue[×1499 ^ "D#\ "E# and "F# are SEM micrographs of cells released from the dorsal vessel after 01 days of culture[ D � ×0499 ^ E � ×1199 ^

F � ×0099[

Fig[ 4[ "A# LM photograph showing endogenous phenoloxidase activity in adult hemocytes[ ×299[ "B# LM photograph showing endogenousphenoloxidase activity in yolk sac!released cells[ ×299[

the fat body to developing oocytes "Kunkel and Nordin\0874 ^ Raikhel and Dhadialla\ 0881# and eventually con!_ned to the yolk sac to sustain embryo development"Fausto et al[\ 0883#[ That vitellogenins may gain accessto embryonic hemocytes was originally suggested byAnderson "0852# in Dacus and by Wolf!Neiss et al[ "0865#in C[ morosus\ while vitellogenin presence in adult hem!

ocytes of Nauphoeta cinerea was reported by Buhlmann"0863#[ In the present study\ a fraction of adult hemocytesand yolk sac!released cells reacted positively when testedimmunohistochemically with an anti!vitellin monoclonalantibody[ These data support the view that vitellin poly!peptides are selectively taken up from the hemolymph invivo or from the culture medium in vitro[

M[ T[ Locci et al[229

Fig[ 5[ "a# SDS!PAGE and "b# Immunoblotting with Mab 01[ Adulthemocytes "H#\ dorsal vessel "V#\ yolk sac!released cells "C#\ yolk sac

envelope "E#[

In summary\ both morphological and immu!nohistochemical observations allow us to conclude thatat least some of the cells released by the yolk sac may beembryonic hemocytes[ Table 0 summarizes some of theproperties shared by adult hemocytes and yolk sac!released cells in C[ morosus[ In early embryonic stages\hemocytes are known to originate from the median meso!derm underneath the yolk mass "Mori\ 0868#[ Duringearly development\ an endodermal cell layer emergingfrom the midgut strands comes to surround the embry!onic yolk mass to eventually give rise to the yolk sac"Ando\ 0851 ^ Anderson\ 0861#[ We hypothesize thatembryonic hemopoietic tissues and:or prospective hem!ocytes may move along the yolk sac wall on theirmigratory pathway to become associated with the dorsalvessel[ The yolk sac may thus act as a temporary hem!opoietic organ in early development\ at least until thedorsal vessel is fully formed[

As to a possibile role hemocytes may play in earlydevelopment\ it is likely that they may be involved invitellin polypeptide processing so as to transport metab!olites from the yolk sac to the developing embryo\ as

Fig[ 6[ "A# LM photograph showing positive reaction with an anti!vitellin monoclonal antibody "Mab 01# in adult hemocytes[ ×299[ "B# LMphotograph showing a positive reaction with an anti!vitellin monoclonal antibody "Mab 01# in cells released from the yolk sac[ ×299[

Table 0[ Properties of adult hemocytes and yolk sac!released cells

Properties Adult Yolk sac!releasedhemocytes cells

Adherence to the substratum yes yesProtrusion of pseudopodia yes yesElectron dense granules yes yesSpindle shaped cells yes yesGlobular cells yes yesPhenoloxidase reaction yes yesReactivity to Mab 01 yes yes

originally suggested by Wolf!Neiss et al[ "0865#[ We arepresently investigating this possibility[

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