SMITHSONIAN MISCELLANEOUS COLLECTIONS
VOLUME 134, NUMBER 6
STUDIES BY PHASE-CONTRAST
MICROSCOPY ON DISTRIBUTION OF
PATTERNS OF HEMOLYMPH
COAGULATION IN INSECTS
(With One Plate)
By
CHARLES GREGOIRE
Department of Biochemistry,
University of Liege, Belgium
IperV
(Publication 4271)
CITY OF WASHINGTON
PUBLISHED BY THE SMITHSONIAN INSTITUTION
MAY 8, 1957
THE LORD BALTIMORE PRESS, INC.
BALTIMORE, MD., U. S. A.
STUDIES BY PHASE-CONTRAST MICROSCOPY
ON DISTRIBUTION OF PATTERNS OF
HEMOLYMPH COAGULATION
IN INSECTS 1
By CHARLES GREGOIRE
Department of Biochemistry,
University of Liege, Belgium
(With One Plate)
A category of hyaline hemocytes (coagulocytes) is playing an im-
portant part in the inception of the plasma coagulation in insect
hemolymph (Gregoire and Florkin, 1950). Differences in the reac-
tions of these corpuscles to contact with foreign surfaces and in those
of the surrounding plasma were recorded previously for various in-
sects (Gregoire, 1951). On the basis of these differences, a classifica-
tion of the process of hemolymph coagulation in insects into four pat-
terns of microscopic pictures has been suggested (Gregoire, 1951).
In former observations on the distribution of the patterns in 420
species of insects, predominance of one of these patterns has been
recorded in several groups of various extension in the classification
(Gregoire, 1955a). The material used in these studies consisted ex-
clusively of representatives of insects from the Old World fauna
(mostly European, and a few Mediterranean and African species).
The aim of the present investigations is to compare the previous
results with data obtained from Neotropical species. In July and
August 1954, during a stay at the Canal Zone Biological Area, the
Smithsonian Institution's tropical preserve on Barro Colorado Island,
the writer collected and examined samples of hemolymph from 630
insects, belonging to about 230 species.
METHODS
The samples of hemolymph were prepared by the procedure used
in former studies (Gregoire, 1951, 1955). In most specimens the
hemolymph issuing from severed or punctured appendages (antennae,
legs, wings, joints of the wing cases) was dropped as rapidly as pos-
sible onto the edge of a cover glass lying on a slide and was allowed
to spread out into films. Under optimal conditions, streaming of the
1 This is No. 7 in the series of papers entitled "Blood Coagulation in Arthro-
pods" published in various journals.
SMITHSONIAN MISCELLANEOUS COLLECTIONS, VOL. 134, NO. 6
Figs. 1-4.—The four tentative microscopical patterns of coagulation (sche-
matic). The drawings have been combined from observations, by means of the
phase-contrast microscope, of about 5,300 samples of hemolymph, in standard
conditions of preparation of the films between slide and coverglass.
Fig. I : Pattern I. Island of coagulation around a hyaline hemocyte (co-
agulocyte). A granular hemocyte and a macronucleocyte of small size are not
involved in the process of coagulation. Extension of the coagulum around the
island and reorganization of the granular clot into meshworks of granular
fibrils have not been represented in the drawing. (Compare with photomicro-
graphs in Gregoire and Florkin, 1950, pis. 1 to 10; Gregoire, 1951, figs. 1-3,
10-12, 14, 27, 28; 1953b, figs. 1, 2, 20, and 22; 1955a, figs. 1-6, 10, 15, 21, 23;
27; and in the present paper, pi. 1, figs. 1, 2, 3, 4, 5, and 7.)
Fig. 2: Pattern II. Extrusion of cytoplasmic expansions by hyaline hemo-
cytes, which appear in the drawing elongated and reduced in size, except three
corpuscles, bulging out at the periphery. The fan-shaped direction of the
cytoplasmic filaments from an incidental bubble is induced in part by currents
in the spreading film of hemolymph. However, this picture has also been
observed to develop slowly, when the hemolymph was at a standstill. Reaction
in the plasma in the shape of glassy elastic veils (no cell fibrin) inside of the
cytoplasmic systems built up by the unstable hemocytes. The veils are fre-
quently detected only by their stretched folds. By pressure exerted on the
coverglass, the structures embedded in the veils move backward and forward,
and preserve their relative positions and distances to each other. The other
categories of hemocytes, agglutinated at random in strands along the highly
adhesive cytoplasmic expansions of the fragile hemocytes, are not represented
on the drawing. (Compare with photomicrographs in Gregoire, 1951, figs. 24-26;
1955a, figs. 17-19, 22, 26, 38, 39, 41 ; and in this paper, pi. 1, fig. 6.)
Fig. 3 : Pattern III. Association of patterns I and II in the same film of
hemolymph : extrusion of cytoplasmic expansions by the hyaline hemocytes, as
in pattern II. Reaction of the plasma consisting of granular veils and of
islands of coagulation. The islands appear within the veils as circular areas of
greater density around the hyaline hemocytes. In this drawing, the reaction
was initiated by alterations in hyaline hemocytes to contact with a foreign body
(hatched). For the fan-shaped disposition of the structures, see above, pat-
tern II. The other categories of hemocytes are not represented on the draw-
ing. (Compare with photomicrographs in Gregoire, 1955a, figs. 11 and 20; and
in this paper, pi. 1, fig. 9.)
Significance of pattern III might be questioned as representing merely a
subsidiary variation of pattern II. However (see legend, plate 1, fig. 9,
Zophobas latticollis Kraatz, Tenebrionidae), pattern III depicts actual differ-
ences appearing consistently in the microscopical picture of coagulation of the
hemolymph, between groups of insects such as Cetoninae (typical pattern II)
and Tenebrionidae (typical pattern III). On the other hand, as already pointed
out (Gregoire, 1951), strong mechanical agencies are apt to give deceptive pic-
tures of pattern III, by inducing extrusion of cytoplasmic expansions from hyaline
hemocytes in insects in which these reactions do not develop spontaneously in
the standard conditions of preparation of the films used for these studies (for
instance, Orthopteroid complex).
Fig. 4: Pattern IV. No visible modification detected by means of the phase-
contrast microscope in the plasma surrounding inert or altered hyaline hemo-
cytes, similar in their appearance to the hyaline hemocytes playing a selective
part in the coagulation of the plasma in the other patterns. (Compare with
photomicrographs in Gregoire, 1951, figs. 29 and 30; 1955a, figs. 12, 13, 14,
and 16.)
NO. 6 HEMOLYMPH COAGULATION IN INSECTS—GREGOIRE
liii
ppfl
Figs. 1-4.
—
(See legend on opposite page.)
4 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. I34
fluid hemolymph reached a standstill before alterations started in the
fragile hyaline hemocytes. The successive steps of the coagulation were
observed by phase-contrast microscopy (Wild M/io). Whenever
possible, several samples were collected from each specimen. In view
of the rapid completion of the process (within a few seconds in many
insects), desiccation did not interfere with the observations, and the
edges of the preparations were not sealed. When the reactions failed
to appear (see pattern IV, below), the preparations were stored in
petri dishes under high moisture and examined subsequently at differ-
ent times. Some degree of evaporation and moderate condensation
of the plasma along the edges of the films were incidentally detected in
those preparations kept for several hours in moistened petri dishes.
RESULTS
DESCRIPTION OF THE PATTERNS OF COAGULATION
The classification of coagulation of insect hemolymph into four
tentative patterns, used in the present study, is essentially based on
differences in the following processes
:
1
.
The irreversible alterations affecting a category of hyaline hemo-
cytes, highly sensitive to contact with solid surfaces, and playing a
selective part in the inception of the coagulation, in contrast with the
other blood corpuscles. In the conditions of phase-contrast micro-
scopy, the unstable hyaline hemocytes appear, especially after their
alterations, as pale, round vesicular elements. Their nucleus is
sharply outlined, relatively small. A few dark granules are scat-
tered in the hyaline cytoplasm. In the other categories of hemocytes
(small stem cells, transitional forms, and various kinds of granular
hemocytes) the nucleus is distinctly larger and the cytoplasm darker.
The cytological differences between the fragile hemocytes and the
other kinds of blood cells have been illustrated in previous papers
(Gregoire and Florkin, 1950; Gregoire, 1951, 1953, 1955a), and ap-
pear in figures 1, 2, 8, and 9 of plate I.
2. The modifications of the plasma, following or accompanying the
alterations in the fragile hyaline hemocytes.
Text figures 1-4 illustrate schematically the microscopical charac-
ters of the four patterns.
Pattern I. Inception of the plasma coagulation in the shape of is-
lands of coagulation around the hyaline hemocytes (text fig. 1 ; pi. 1,
figs. 1, 2, 3, 4, 5, and 7).—Selective alterations in the unstable hyaline
hemocytes result in exudation or in explosive discharge of cell ma-
terial into the surrounding fluid. Coagulation of the plasma starts in
NO. 6 HEMOLYMPH COAGULATION IN INSECTS GREGOIRE 5
the shape of circular islands of granular consistency around the al-
tered hyaline hemocytes. The islands of coagulation develop to a
certain size, with individual and specific variations, then their increase
stops. At the beginning of the process, the islands are scattered and
separated by fluid channels. When the coagulation proceeds farther,
the plasma in these channels clots into a granular substance in which
the islands preserve generally their original size and shape. General
solidification of the film may occur. The coagulum, of granular ap-
pearance, is progressively modified into delicate meshworks of
granular fibrils.
Pattern II. Extrusion of cytoplasmic expansions by hyaline hemo-
cytes, with development of cytoplasmic meshworks. Reaction in the
plasma in the shape of veils (text fig. 2 ; pi. 1, fig. 6).—On contacting
the glass, a category of fragile hyaline hemocytes undergo alterations
that differ from those observed in pattern I. These elements extrude
threadlike cytoplasmic expansions, which may reach a great length.
These expansions exhibit intense thigmotropism toward solid par-
ticles, other hemocytes, and physical interfaces (bubbles). These al-
terations result in constitution of cytoplasmic meshworks of various
complexity, on which the other kinds of hemocytes are passively
agglutinated.
The reaction in the plasma after these cellular changes occurs in
the shape of transparent, elastic, and contractile veils, developed
within the cytoplasmic systems built up by the hyaline hemocytes, or
in their vicinity. The other categories of hemocytes do not take part
in the formation of these cytoplasmic meshworks. They are passively
agglutinated along the highly adhesive cytoplasmic filaments sent out
by the hyaline hemocytes and are subsequently embedded with them
in the plasma veils.
In several insects the alterations in the unstable hemocytes are not
followed by changes in the plasma and the modifications of the hemo-
lymph in vitro consist only of a cellular reaction.
Pattern III. Patterns I and II combined (text fig. 3 ; pi. 1, fig.
9).—The microscopical picture of pattern III consists of an asso-
ciation of the reactions described above in patterns I and II. In the
same film of hemolymph, hyaline hemocytes produce cytoplasmic
expansions (pattern II) while islands of coagulation (pattern I) de-
velop around the body of these corpuscles. The islands are either
isolated or appear as denser areas within the veils characterizing the
reaction in the plasma in pattern II.
Pattern IV. No modification in the hyaline hemocytes, or altera-
tions not followed by visible reaction in the plasma (text fig. 4) .
—
In
6 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. I34
the hemolymph of various insects, hemocytes resembling in their
cytological characters the fragile corpuscles involved in the other
patterns do not visibly alter. They appear as large, pale vesicles in
which a few dark particles are scattered. In several insects these
elements are the remnants of darker refractile hyaline hemocytes,
which undergo clarification after explosive discharge of a part of
their cytoplasm. In the vicinity of these inert or altered hyaline hemo-
cytes no change can be detected under the phase-contrast microscope
in the consistency of the plasma, which remains permanently fluid.
DISTRIBUTION OF THE PATTERNS OF COAGULATION IN THE DIFFERENT
GROUPS OF INSECTS INVESTIGATED
In the table below, the names of the species are followed by the
numbers of specimens studied (adults, unless otherwise stated) and
by the patterns of coagulation provisionally found predominant or
representative on the basis of the microscopical study of several
samples of hemolymph obtained from these specimens. Incidental
findings of other patterns are also reported under "Comments."
In several taxonomic groups, the average pattern recorded on cor-
responding material from the Old World previously studied (Greg-
oire, 1955a; Gregoire and Jolivet, unpublished) follows the list of the
Neotropical material in the table.
The patterns of coagulation described above have been represented
in the table by the following symbols
:
® : pattern I : inception of the plasma coagulation in the shape of
islands of coagulation around hyaline hemocytes.
O : pattern II : development of cytoplasmic meshworks by hyaline
hemocytes. Reaction in the plasma in the shape of veils.
0-: pattern II: incomplete. Emission of cytoplasmic expansions,
characterizing the reactions of the hyaline hemocytes in pat-
tern II, but unaccompanied by formation of veils in plasma.
(•) : pattern III : patterns I and II combined.
—
: pattern IV: no visible coagulation.
( ) : pattern incidentally or exceptionally recorded in limited fields
of preparations exhibiting predominantly another pattern.
( ?) : microscopical characters of a pattern not clear-cut or equivocal.
Other abbreviations used : sp., species ; spm., specimen.
Gradations in the intensity of the reactions, especially with regard
to pattern I, are indicated by the following symbols : I poor (scarce
fringes of clotted plasma around the altered hyaline hemocytes, with-
NO. 6 HEMOLYMPH COAGULATION IN INSECTS—GREGOIRE 7
out extension of the coagulation; I (scattered islands of coagulation
of various sizes, with moderate coagulation of the fluid in the chan-
nels) ; I*, I**, I*** (islands around all the hyaline hemocytes, sub-
stantial and general coagulation. In I***, the films appear to the
naked eye with a bluish opalescent color).
MICROSCOPY (PARTICULAR REACTIONS)
Orthopteroid Complex.—Pattern I has been uniformly recorded
in all the insects of the orthopteroid complex listed above. However,
intensity in the reaction differed in the various groups : in this respect,
Blattodea and Gryllidae exhibited the most substantial coagulation.
These groups were followed, in order of decreasing intensity, by
Mantodea, Phasmoptera, Tettigoniidae, and Acrididae.
In the present material, alterations in plasma appearing around
hemocytes other than the fragile hyaline hemocytes were detected
around a few macronucleocytes of small size (stem cells) in samples
from Paroecanthus podagrosus and from Phasma sp. As already
pointed out (Gregoire, 195 1, 1955a) such reactions are exceptional.
Heteroptera.—As shown in the list, absence of visible change in
the plasma was the predominant picture in all the specimens of the
present material. The few modifications that might suggest subsidiary
participation of a pattern other than pattern IV were equivocal.
In Ghilianella sp., Triatoma dimidiata, Rhiginia sp., Apiomerus
ochropterus (Reduviidae), Mecisthorhinus marmoratus (Pentatomi-
dae), dark oval hyaline hemocytes, surrounded by a refractile halo,
underwent sudden clarification after explosive ejection of cytoplasmic
substance into the surrounding fluid. These discharges did not bring
about changes in the consistency of the plasma. Similar alterations
have been described previously (Gregoire, 1955a) in various insects,
especially in lepidopteran and dipteran larvae and in dipteran adults.
In several samples from different families, granular precipitates,
unrelated to the presence of hemocytes in the vicinity, were found in
films of hemolymph around air bubbles and along the edge of the
coverglass. Though the preparations were maintained between the
observations in petri dishes under high moisture, these modifications
resulted probably from a slight degree of evaporation and conden-
sation at the periphery of the films. Pressure exerted on these
precipitates dispersed granular particles ; this reaction is different from
that taking place under similar mechanical agencies, in an actual
coagulum, in which extension of the granular and fibrillar structures
is followed by elastic contraction without dissociation.
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NO. 6 HEMOLYMPH COAGULATION IN INSECTS GREGOIRE 2"]
Homoptera.—Pattern I, with general solidification of the plasma,
was consistently observed in Cicadidae, Cicadellidae and Fulgoridae.
In the last family {Laternaria, Phrictus), coagulation of the hemo-
lymph was especially substantial ; the films of hemolymph were in-
stantaneously transformed into opalescent bluish clots, embedding all
the hemocytes (altered fragile hemocytes, numerous small macro-
nucleocytes, and transitional forms to various types of granular hemo-
cytes).
Coleoptera.—The various groups of Coleoptera listed in the table
exhibited a great diversity in the reactions of their hemolymph in
vitro. However, predominance of one of the patterns characterized
several groups.
Dark hyaline hemocytes, undergoing clarification after discharge of
substance (see Heteroptera above, and Gregoire, 1955a, discussion,
p. 129), were observed in Agra sp. (Carabidae) and in Veturius
platyrrhinns (Passalidae).
The reactions detected in Scarabaeidae (especially Melolonthinae,
Rutelinae, Dynastinae) were essentially identical to those reported
previously as representative of this family. Upon withdrawal, the
hemolymph became immediately viscous and ropy. The hyaline
hemocytes, relatively numerous (64 percent of the total hemogram
in Lagochile sparsa Ohaus) and of small size, extruded spontaneously
cytoplasmic expansions, soon embedded, like the other hemocytes, in
the veil-like reaction developing in the plasma (pi. 1, fig. 6).
In Zophobas latticollis Kraatz, pattern III, characterizing several
species of Tenebrionidae, developed with a special clarity : cytoplasmic
expansions of the hyaline hemocytes and transparent glassy veils
(pattern II) appeared immediately upon withdrawal of the hemo-
lymph. The consistency of the veils became granular, while circular
areas of greater density (islands of coagulation: pattern I) grew out
around several hyaline hemocytes already involved in the constitu-
tion of cytoplasmic systems (pi. 1, fig. 9).
In the specimens of Sandalidae, the film of hemolymph consisted
of a substantial syrupy granulum embedding tiny nuclei of altered
unidentifiable hemocytes. The pattern of coagulation could not be
safely established in these specimens.
In Lampyridae, dense suspensions of particles normally present in
the hemolymph of these insects, as in other groups (Coccinellidae,
various Chrysomelidae), interfered with the detection of the pattern
of coagulation.
Among Cerambycidae, subfamily Prioninae {Stenodontes, Calli-
28 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. I34
pogon) exhibited one of the most substantial coagulations recorded
among all the insects examined in this and in the previous studies.
Hymenoptera.—Rapid collection of the hemolymph without con-
tamination with foreign tissues was difficult in small specimens and
in dry ones. In ants, large numbers of specimens were used, and the
only samples not discarded were those in which a limpid drop of
hemolymph could be collected and a rapid spreading out of the films
performed.
In view of the scarcity of the species available and the large inter-
specific and intraspecific variations in the reactions observed in this
order of insects, the predominant patterns could not be established
safely for several species and groups. Some patterns actually recorded
in a part of the samples correspond possibly to incomplete reactions.
Pattern I was observed in all the samples collected from all the
females, males, and workers of Paraponera clavata (Formicidae) and
Chlorion (Sphecidae), and pattern III in the three specimens of
Mutillidae captured.
In larvae of Camponotus sericeiventris, numerous hemocytes were
loaded with refractile inclusions, and no modification of the plasma
appeared in that material.
The Hymenoptera listed in the table were characterized by the small
size of their hemocytes and of the islands of coagulation around the
hyaline hemocytes, even in the samples in which a substantial coagu-
lation was recorded. In the latter preparations, a considerable ex-
tension of the coagulation took place from around the islands of
coagulation, which appeared in the granular clots as small circular
areas of greater density, centered by the fragile hyaline hemocytes
and remaining distinct in the general coagulation of the plasma (pi. I,
%s. 3, 4, 5).
Lepidoptera (larvae).—In the two specimens of lepidopteran lar-
vae, the reactions of the hemolymph in vitro were identical to those
described and illustrated elsewhere in a large number of caterpillars
(Gregoire, 1955, pp. 118-120 and pis. IX and X: pattern II, with
large individual variations in the completion of the process, fre-
quently incomplete, as in the two specimens listed in the table). In the
films of hemolymph, refractile hyaline hemocytes underwent clarifi-
cation after rupture of the cell boundaries and discharge of substance,
as illustrated in figures 42-50 of the above-cited paper.
DISCUSSION
1. The four patterns used in the present study are an attempt to
classify the disparities recorded in insects with regard to the micro-
NO. 6 HEMOLYMPH COAGULATION IN INSECTS—GREGOIRE 29
scopical picture of films of clotting hemolymph, observed by phase-
contrast microscopy in standard conditions of preparation.
Objection that these patterns might result from random artifacts
has been examined elsewhere (Gregoire, 1955a).
In control observations, the clotting process was compared in films
of hemolymph spread out under glass by the standard procedure and
in clot plugs spontaneously formed at the wound site and gently
squeezed under glass after completion of the process. In both in-
stances, the microscopical alterations characterizing the same pattern
were recorded. The standard conditions of preparation of the samples
of hemolymph seem therefore to be a faithful reproduction of the
alterations occurring during the undisturbed natural process.
2. Whatever each pattern might signify at the cytological 2 or bio-
chemical level, 3 most results of the present and other studies 4 suggest
2 Among the factors implied in the process of coagulation, the patterns reflect
actual inequalities between species and higher taxonomic groups in the degree
of sensitiveness to contact with solid surfaces of the fragile hyaline hemocytes
selectively involved in the inception of the coagulation, in the nature of the
alterations undergone by these unstable cells, and in the rapidity with which
these alterations develop. These differences affect the subsequent reaction of
the plasma. As shown in the present and in previous studies, a similar degree
in sensitiveness of the fragile hemocytes is frequently shared by insects belong-
ing to a same group.
A tentative identification of the fragile hyaline hemocytes has been reported
elsewhere (Gregoire, 1953a; 1955a, p. 129). A part of these corpuscles exhibits
cytological features in common with the oenocytoids. In several groups (Odo-
nata, Hemiptera-Heteroptera, various species of Coleoptera, lepidopteran and
dipteran larvae, Trichoptera, and some Hymenoptera) these corpuscles appeared
in the films of hemolymph in the shape of highly refractive or dark hyaline
hemocytes, which undergo clarification after explosive discharge of substance.
The same corpuscles differ, however, in other characters from the classical
description of the oenocytoids (1955a, discussion, p. 131). On the other hand, the
fragile hemocytes selectively involved in coagulation are referred to by Jones
(1954) as cystocytes, in Tenebrio molitor.
3 The scarcity of the data available at the present time does not enable one
to establish whether actual biochemical differences characterize each of the
four patterns, and especially the two aspects presented by the reactions in the
plasma, the granular substance (in the islands of coagulation and in the areas
of extension: pattern I), and transparent glassy veils (pattern II). As sug-
gested by observations of films of varying thickness, it is unlikely, as reported
elsewhere (Gregoire, 1955a, discussion, p. 128) that the twofold aspect of the
plasma changes is related merely to differences in concentration or in thickness of
the clotted films. The veils are not to be identified with the products of general
disintegration of the hemocytes (cell fibrin).
An adequate test of the validity of the patterns would be to determine
whether biochemical differences correspond to microscopical pictures as dif-
ferent as those consistently recorded, for instance, in insects belonging to the
30 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 134
that the patterns are not individual particularities, except in a few
equivocal cases. 6 The patterns rather characterize species, more fre-
quently taxonomic groups (genera, families, suborders, or orders).
Repeated samplings of hemolymph collected from several speci-
mens of the same species, or from different species belonging to the
same higher taxonomic category, made it possible to record consis-
tently the same pattern in groups of various taxonomic importance
such as the Orthopteroid complex (pattern I),
6 several families of
Heteroptera (especially Reduviidae, Coreidae, Pentatomidae) (pat-
tern IV), Belostomatidae and Nepidae (pattern I), three families of
Homoptera (Cicadidae, Fulgoridae, Cicadellidae) (pattern I), among
Coleoptera, Hydrophilidae (pattern IV), Staphylinidae (pattern IV),
several subfamilies of Scarabaeidae (Rutelinae, Melolonthinae, Dy-
nastinae, Geotrupinae, Trichiinae and Cetoninae) (pattern II),
Heteromera (Tenebrionidae, Lagriidae, Monommidae, Oedemeridae
and Meloidae; patterns I and III), Cerambycidae (pattern I), Cur-
culionidae (pattern IV), several families of Lepidoptera (larvae;
pattern II), Tenthredinidae (patterns I and III).
3. Other groups (Cicindelidae, Carabidae, Dytiscidae, Silphidae,
Passalidae, Coprinae, Elateridae) exhibited large intraspecific and
interspecific variations in the patterns of coagulation recorded. In
view of the diversity of the reactions in these groups, the pattern
representative or predominant could not be established with certainty.
However, at the genus level predominance of a pattern appeared in
genera such as Carabus (pattern III), Agra (pattern I), Hydaticus
(pattern IV), Dytiscus (pattern III) Cybister (pattern I), Necro-
phorus (pattern I).
4. In the homogeneous groups listed above, the Neotropical ma-
terial and the insects from the Old World supplied identical results
orthopteroid complex (pattern I), in insects from several families of Scara-
baeidae (Rutelinae, Melolonthinae, Dynastinae, Cetoninae) (pattern II), He-
teromera (pattern III), and other groups of insects in which no visible modi-
fication could be detected in the plasma (e.g., Staphylinidae, Hydrophilidae, and
many Heteroptera) (pattern IV) under the phase-contrast microscope.
4 Gregoire ( 1 95 1 , 1955); Gregoire and Jolivet (unpublished). The total
material investigated consists of approximately 5,300 samples of hemolymph,
collected from 3,400 specimens belonging to about 850 species.
5 In most of these cases, the scarcity of the material available suggests that
individual variations, incomplete reactions, or accidental artifacts (mechanical
agencies; see Gregoire 1955a, p. 124(1.) might confuse the actual pattern.
6 In the highly homogeneous orthopteroid complex, differences in the inten-
sity of the clotting reaction could be detected between several groups (see
table).
NO. 6 HEMOLYMPH COAGULATION IN INSECTS GREGOIRE 31
with regard to the pattern of coagulation predominant or representa-
tive of the taxonomic category.
Such consistency suggests that the patterns of hemolymph coagu-
lation are a character of taxonomic significance (in a broad sense).
Whether that type of character is of more or less applicability in
phylogenetical controversies, is a question left to competent phylo-
geneticists. It might, however, be stressed that the process of hemo-
lymph coagulation is in no way related directly to any type of struc-
tural or ethological criteria commonly used for defining and grouping
taxonomic categories. It is therefore of interest to check tentatively
some taxonomic relationships on the basis of the presented data.
5. As pointed out elsewhere (Gregoire, 1955a, pp. 136-137), ran-
dom coincidence does not seem to be entirely responsible for explain-
ing some correlations between phylogenetic position of certain groups
of insects and microscopical aspect of the coagulation of their hemo-
lymph. In this respect, the Neotropical material examined here sup-
ports former tentative suggestions concerning most of these corre-
lations. 7
Pattern I has been heretofore uniformly recorded in Blattodea and
in the other groups ranged within the orthopteroid complex. The
mechanism involved in this pattern is identical to one of the types of
coagulation described by Hardy (1892), Tait (1910, 1911), Tait and
Gunn (1918), Numanoi (1938), and Gregoire (1955b) in crustacean
blood, in which a special category of cells, the Hardy's explosive cor-
puscles, corresponding to the insect hyaline hemocytes or coagulo-
cytes (Gregoire and Florkin, 1950), play a selective part in the in-
ception of the coagulation of the plasma.
Pattern I has also been recorded among various unrelated groups
of insects, especially in groups characterized by the retention of
various primitive characters, such as the Homoptera. In this respect,
the present study has brought information on groups not represented
in the material previously investigated.
From these data, pattern I might be considered as a generalized
primitive mechanism of coagulation of insect hemolymph.
The mechanism of coagulation illustrated in pattern II has been
observed, unmixed or predominant, in relatively recent groups of
7 After the completion of this paper, the patterns of coagulation were recorded
in 400 insects collected in September 1956 at Tingo Maria (Peru) and in Octo-
ber 1956 on Barro Colorado Island. The results are in agreement with those
reported here, with regard to the predominance of one of the patterns in the
following groups : Orthopteroid complex, Hemiptera, Homoptera, Scarabaeidae,
Tenebrionidae, Cerambycidae, Curculionidae, Vespidae, and Diptera.
32 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. I34
insects, such as Scarabaeidae (except Coprinae—see next paragraph)
and lepidopteran larvae. As shown in the table, the present investi-
gations have confirmed the predominance of the patterns previously
reported for these groups.
6. In the samples of Neotropical species of Passalidae and of
Coprinae, the islands of coagulation characterizing pattern I were
absent or exceptionally recorded in the samples, while in the African
specimens studied until now (Gregoire and jolivet, unpublished),
these islands of coagulation appeared frequently or consistently in
many samples. In view of the scarcity of the material and the diversity
in the reactions characterizing these two groups, random variations
might be responsible for these divergences between the results.
SUMMARY
Coagulation of the hemolymph in vitro has been investigated by
phase-contrast microscopy in 630 specimens from 230 Neotropical
species of insects. The present material includes samples of hemo-
lymph from insects belonging to 17 families not represented in previ-
ous related studies.
A tentative classification of the process of coagulation into four
patterns, suggested previously, has been used, and the patterns charac-
terizing provisionally each species have been determined and reported
in tabular form.
The Neotropical material and the data collected formerly on species
from the Old World (altogether approximately 850 species), sup-
plied consistent results with regard to the predominance of some of
the patterns in several taxonomic groups of various extension in the
classification.
In a condensed form, the investigations on the distribution of the
patterns of hemolymph coagulation in the different orders have shown
:
(1) In the Orthopteroid Complex, a great uniformity of reaction, in
the shape of pattern I, possibly a generalized primitive mechanism of
coagulation of the hemolymph
; (2) in several families of Heteroptera,
absence of a visible reaction in plasma (pattern IV), in striking con-
trast to two families of the same order, Belostomatidae and Nepidae,
which exhibited a substantial coagulation (pattern I)
; (3) in Homop-
tera, a substantial reaction in the shape of pattern I, representative or
predominant, in Cicadidae, Fulgoridae, and Cicadellidae
; (4) in Co-
leoptera, as a taxonomic group, a large heterogeneity in the reactions.
However, in this order, uniformity of reaction or predominance of a
pattern was detected at the infraorder level, especially in Hydrophili-
NO. 6 HEMOLYMPH COAGULATION IN INSECTS GREGOIRE 33
dae (pattern IV), Staphylinidae (pattern IV), Scarabaeidae (pattern
II, with the exception of Coprinae: patterns I and III), Heteromera
(patterns I and/or III), Cerambycidae (pattern I), Curculionidae
(pattern IV), and in a few genera reported in the table ; (5) in Lepi-
doptera (larvae), predominance of pattern II, with the possible ex-
ception of Saturniidae (pattern III)
; (6) in Hymenoptera, occur-
rence of patterns I and III in several taxonomic groups, in Apidae,
scarce coagulation or absence of plasma reaction.
ACKNOWLEDGMENTS
I am greatly indebted to James Zetek, former Resident Manager
of the Canal Zone Biological Area, and Mrs. A. Gomez, secretary
at the station, who did everything possible to facilitate my work dur-
ing my stay at the laboratory on Barro Colorado Island. I acknowl-
edge with sincere appreciation the assistance of Paul Swift, chief of
the Eastman Kodak Tropical Research Laboratory, Panama City, who
developed the photographic plates, and Messrs. Bocanegra and Ruiz
who supplied me with several specimens.
I wish to thank Dr. Remington Kellogg, Director of the United
States National Museum and Dr. Waldo L. Schmitt, Head Curator
of the Department of Zoology, for authorization to have the material
determined in the Museum, and to express my gratitude to the fol-
lowing for identification of the specimens: Dr. M. Beier, Dr. R. E.
Blackwelder, O. L. Cartwright, Prof. L. Chopard, G. Fagel, Prof.
W. D. Hincks, Ch. Jeuniaux, K. V. Krombein, M. N. Magis, C. F. W.
Muesebeck, Miss S. Parfin, Miss L. Russell, Dr. R. I. Sailer, Dr.
M. W. Sanderson, Dr. M. R. Smith, Dr. Thomas E. Snyder, T. J.
Spilman, Geo. B. Vogt, Miss R. E. Warner, Dr. N. Weber, Dr. C.
Willemse, Dr. W. W. Wirth, and Dr. D. A. Young. Special thanks
are due O. L. Cartwright, Acting Curator of the Division of Insects,
United States National Museum, for collecting and mailing the data
;
Dr. J. Leclercq for much information about taxonomic problems
related to the present paper ; and the Patrimoine de l'Universite de
Liege, for defraying the expenses of shipment of the microscope and
equipment used in the present studies.
34 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. I34
REFERENCES
The literature on hemolymph coagulation in insects has been reviewed in the
papers cited below under Gregoire, 1951, 1953a, and 1955a.
Gregoire, Ch.
1951. Blood coagulation in arthropods. II. Phase contrast microscopic
observations on hemolymph coagulation in sixty-one species of
insects. Blood, vol. 6, pp. 1173-1198.
1953a. Coagulation de l'hemolymphe chez les insects hemipteroides. Arch.
Internat. Physiol., vol. 61, pp. 237-239.
1953b. Blood coagulation in arthropods. III. Reactions of insect hemolymph
to coagulation inhibitors of vertebrate blood. Biol. Bull., vol. 104,
PP- 372-393.
!953c Sur la coagulation de l'hemolymphe des termites. Arch. Internat.
Physiol., vol. 61, pp. 391-393-
1954. Sur la coagulation de l'hemolymphe des termites (deuxieme note).
Arch. Internat. Physiol., vol. 62, pp. 117-119.
1955a. Blood coagulation in arthropods. V. Studies on hemolymph coagu-
lation in 420 species of insects. Arch. Biol., vol. 66, pp. 103-148.
1955b. Blood coagulation in arthropods. VI. A study by phase-contrast
microscopy of blood reactions in vitro in Onychophora and in
various groups of arthropods. Arch. Biol., vol. 66, pp. 489-508.
Gregoire, Ch. and Florkin, M.
1950. Blood coagulation in arthropods. I. The coagulation of insect blood,
as studied with the phase contrast microscope. Physiol. Comp.
et Oecol., vol. 2, pp. 126-139.
Hardy, W. B.
1892. The blood corpuscles of the Crustacea, together with a suggestion as
to the origin of the crustacean fibrin-ferment. Journ. Physiol.,
vol. 3, pp. 165-190.
Jones, J. C.
1954. A study of mealworm hemocytes with phase contrast microscopy.
Ann. Ent. Soc. Amer., vol. 47, pp. 308-315.
Numanoi, H.
1938. On crustacean blood coagulation. Japan. Journ. Zool., vol. 7, pp.
613-641.
Tait, J.
1 910. Crustacean blood coagulation as studied in the Arthrostraca. Quart.
Journ. Exper. Physiol., vol. 3, pp. 1-20.
191 1. Types of crustacean blood coagulation. Journ. Mar. Biol. Assoc,
vol. 9, pp. 191-198.
Tait, J., and Gunn, J. D.
1 91 8. The blood of Astacus fluviatilis: a study in crustacean blood, with
special reference to coagulation and phagocytosis. Quart. Journ.
Exper. Physiol., vol. 12, pp. 35-80.
NO. 6 HEMOLYMPH COAGULATION IN INSECTS—GREGOIRE 35
EXPLANATION OF PLATE 1
Films of hemolymph spread out between slide and coverglass, immediately
upon shedding from severed appendages. Phase-contrast microscope (Wild
M/10). Scale: 20 microns.
Figs. 1 and 2. Stenodontes (Mallodon) molarius Bates (Cerambycidae, Pri-
oninae). (Pattern I***: very substantial coagulation.) All the hyaline
hemocytes are surrounded by islands of coagulation. Considerable ex-
tension of the granular coagulum. In figure i, three elements belonging
to other categories of hemocytes are passively embedded in the clot.
Figs. 3, 4, 5. Paraponera clavata (Fabricius) (Formicidae). (Pattern I ***).
Islands of coagulation of small size. Extension of the coagulum. In
figure 3, a small hyaline hemocyte (on the right) in the center of a small
island of coagulation. On the left, two granular corpuscles embedded in
the clot.
Fig. 6. Cetoninae sp. (Scarabaeidae). (Pattern II). Typical reaction of the
hemolymph in vitro, as it appears in several subfamilies of this group (see
table). Many hyaline hemocytes of small size with their cytoplasmic
expansions are embedded in a substantial veil. Identical pictures were ob-
served in several species listed in the table, especially in Lagochile, Pelid-
nota, Phalangogonia, Trisogeniates, Aspidolea, Cyclocephala, and Dys-
cinetus.
Fig. 7. Phrictus quinquepartihis Distant (Llomoptera, Fulgoridae). (Pattern
I***). Two hyaline hemocytes, each surrounded by an island of coagula-
tion. Considerable extension of the coagulum between the islands, which
preserve their size and shape.
Fig. 8. Pseudophyllidae (Tettigoniidae). Larva, first stage, male (Pattern I).
The process of coagulation was slow and developed poorly in this specimen.
When the picture was recorded, no reaction had yet developed in the plasma
around the hyaline hemocyte shown in the center, between two other blood
elements, including a granular hemocyte.
Fig. 9. Tenebrionidae sp. Picture representative of pattern III, predominant in
this family (see description of the coagulation in Zophobas latticollis
Kraatz, in the text, p. 27). Fan-shaped disposition of the threadlike cyto-
plasmic expansions of hyaline hemocytes, diverging from an air bubble on
which these highly adhesive structures are anchored. Plasma reaction in
the shape of a veil, with denser areas around hyaline hemocytes, correspond-
ing to islands of coagulation. (Compare with Gregoire, 1955a, figs. 11
and 20. See legend of text figs. 2 and 3.)

SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 134, NO. 6, PL. 1
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(See explanation at end of text.