ABSTRACT. The growth of primary cultures of honey bee (Apis mellifera L.) haemocytes and the evaluation of several
parameters and of the growth conditions were aimed at. Use was made of haemolymph extracted from variously aged (4-7
days) larvae of working bees. The direct examination under the inverted microscope and Giemsa staining were used for the
morphological characterisation, and the vital exclusion technique with a 0.5% trypan blue solution was used for the percental
evaluation of the haemocytes viability. The examinations were conducted every two days starting with day 5 of culturing up to
day l4 in the case of the cultures from 4-5 day old larvae, and on days 14, 18 and 21 of cultivation in the case of those from
larvae aged 6-7 days. Primary haemocytes cultures (+_ fat cell fragments) from working bees larvae aged 4-5 and 6-7 days
were obtained. Both the haemocytes (qranulocytes + oenocytoids) and the cells of the fat body fragments (fat cells +
oenocytes) were morphologically characterised and identified; also, the viability of granulocytes + oenocytoids was evaluated
for the period of 5-2l days of cells culturing.
KEY WORDS: Haemocytes, in vitro growth, honey bee larvae
There exists a very small number of papers on the cultivation in vitro of the cells and tissues of honey bee (Apis mellfera
L.). In 1968, Stanley  and in 1971, Giauffret [I] reported the growth of primary cultures of queen pupae ovarian tissue,
and in 1988, Van Steenkiste  that of primary haemocytes cultures.
The in vitro haemocytes cultivation is useful in the study of the cell mechanisms of the bees immunity . To this effect, we
consider that the possibility to obtain primary haemocytes cultures has to be first evaluated, and then the parameters and
the survival conditions for the haemocytes in the culture have to be determined, followed by trials for stabilising the primary
culture (meaning the obtainment of a haemocyte cell line) to thus create an important tool for the study both of phagocytosis
and the nodules formation, and of the immune proteins synthesis inducement.
'fo this aim, this paper had in view the obtention of primary haemocytes cultures and the evaluation of several parameters
(morphology, density, viability, presence of other cellular types) and of the growth conditions.
MATERIAL AND METHOD
Honey comb fragments with variously aged working bee larvae were collected from a healthy bee family. Some of the 4-5
days old larvae (L 4-5) were processed the same day. The other honey comb fragments were kept at 30°C for 48 hours,
the larvae reaching the age of 6-7 days (L 6-7) being processed.
In vitro haemocytes growth
The working protocol is adapted after Van Steenkiste  and Giauffret [1 ]. The larvae were decontaminated by immersion
into 70% ethyl alcohol for 1 minute and into an antibiotics solution (penicillin 500 IU/ml, streptomycin 500 ug/ml, amphotericin
B 2.5 ug/ml) in MEM Eagle (Gibco-BRL) for 30 seconds, followed by drying against filtre paper. The haemolymph was
extracted by means of Pasteur pipettes from the dorsal vessel of the abdominal segments VIII-IX where the vessel diameter
is maximum [Nelson, 1924 in 2]. The cultures were grown on plastic 24-welled Linbro plates (Flow Laboratories). 300 ul of
medium Grace (Sigma), pH 6.5 supplemented with 4% lactalbumin hydrolysate (Sigma). 5% bovine foetal serum (Sigma) and
antibiotics (penicillin 50 IU/ml, streptomycin 50 ug/ml), and also added with the haemolymph extracted from 1-2 larvae were
distributed into each well. Additionally, as the haemolymph from some larvae contained small fragments of fat body as well,
the wells with the culture had to be divided into wells with haemolymph (H) and those with haemolymph + fat body
fragments (H+FB). After 15-45 minutes, 250 ul of the medium in each well were replaced with 950 ul/well, the final culture
volume being 1000 ul. The plates were adhesive taped, placed - together with a piece of wet paper napkin - into a plastic
bag , and incubated at an initial and a final temperature of 25.5 and of 28.50 C respectively in a normal atmosphere.
Culture parameters evaluation
The morphological characterisation was by the direct examination under an inverted microscope (30x, 100x, 200x) using an
ocular micrometer as well, and by the modified Giemsa staining,  of the preparations of the cell suspension extracted
from the wells.
The viability of the haemocytes, that is granulocytes + oenocytoids (GR + OE), was evaluated % by the vital exclusion
technique using a 0.5% trypan blue solution [6; Yip, Anersperg, 1973 in 7]. The viability was evaluated in both wells H and
wells H + FB.
These examinations were conducted every two days, starting with day 5 up to day 14 of culturing in the case of the
cultures from L 4-5, and on days 14, 18 and 21 of culturing in the case of the L 6-7 cultures.
RESULT AND DISCUSSIONS
One hour post cultivation many small (10-17 um in diameter) round cells with a bright heterochromous cytoplasm, as well as
rare, elongated, polymorphous, tibroblast-like cells adherent to the well bottom were noticed by direct examination under the
microscope. In wells H + FB, large (40-70 Ftm in diameter) opaque cells with a muriform aspect, as well as rare cells of
intermediate dimensions (20-30 um in diameter) were also noticed, both cell categories being spherical.
48 hours post cultivation, the fibroblast-like cells number in wells H dropped (Figures 2, 3 and 5). the large majority of the
cells being represented by the small spherical ones that have a marked heterochromia (cytoplasmatic granules or vesicles)
and a visible cytoplasmatic membrane. The large (2-3 um in diameter) and clearly delimited cptoplasmatic granules/vesicles
have a bright content (Figures 2 and 5). In the Giemsa stained preparations, these cells were doubtelessly identified as
belonging to the GR'-0E group in keeping with the previously mentioned criteria ; this is easy to explain through the mere
fact that the larvae have the mean procental value of 96% . Van Steenkiste  found only GR in the haemocytes cultures
from L 4 and L 5. Besides GR + OF, the large and the intermediate cells may rarely be noticed as well (Figure 4).
The number of GR + OE is smaller in wells H + FB than in wells H, the large cells being, predominant. The latter have a
marked heterochromia and two areas inside: a central, denser area of a muriform aspect and not clearly delimited, that is
the nucleus 13-20 um in diameter surrounded by intimately adhered vesicles, a peripheral ring-shaped and more transparent
area containing many granules/vesicles having characters similar to those of GR + OE but larger (3-10 um in diameter) as
seen in Figures 6 and 7. The cytoplasmatic membrane of most of the cells is visible intact and smooth; there also exist,
however, cells with a discontinuous membrane and granules under dispersal (Figures 6 and 7) These large cells were
identified-based on the morphological characters described - as fat cells. The type of the intermediately-sized cell is the
most rarely found; it is round with a relatively large nucleus (10-13 um in diameter), a ringshaped cytoplasm, a fine
heterochromia, and smaller granules/vesicles than those of the fat cells and GR I OE (below 2 um in diameter) that are
concentrated towards the nucleus. Its cell membrane is bright, visible and clearly delimited (Figures 4 and 7); the nuclear
area is darker, while the peripheral one is brighter. This cell type was identified as an oenocyte.
During the 48 hours -21 days time interval, the GR + OE were found to generally maintain their afore enumerated
characters; however, after 14 days of growth, the cytoplasmatic membrane starts to wrinkle, the granules/vesicles get
crowded towards the periphery. Sometimes the granules/vesicles become proeminent (probably under going exocytosis)
so that the cells become muriform, staying however alive. Also, the proportion of the disintegrated fat cells rises, these
cells being reduced to a small number of granules/vesicles attached to the nucleus rest being dispersed into the medium.
Moreover, tile oenocytes membrane becomes more and more difficult to observe. The cells probably entering a process of
The viability of the L 45 GR + OE varied, during the 5-14 days period, from 81.1% (on day 5) to 90.2% (on day 9), while that
of the L 6-7 GR + OE ranged, during the 14-21 days period, between 90.4% (on day 14) and 60.7% (on day 21). The
viability values recorded with the L 4-5 and L 6-7 GR + OF cultures are presented in Table I and in Figure 1 in comparison
with those found by Van Steenkiste with the I. 5 GR at 25"C.
Following an upward viability course up to day 9, a slight decrease was recorded up to day 12, with a plateau value up to
day 14 (in the case of the L 4-5 GR + OE tile examination was stopped on day 14 on account of the culture getting
infected). As for the L 6-7 GR + OF, the viability was examined only on days 14, 18 and 21 since cultivation: following the
registration of a value very close to that of the L 4-5 haemocytes after 14 days, a slight decrease was recorded on day 18,
followed by a marked one on day 21. The examination of the L 6-7 culture was discontinued as the number of wells was
Figure I shows the L 4-> GR + OE survival values recorded by us for tile 5-14 days interval to be somewhat smaller - by up
to 16% (on day 5) - than those obtained by Van Steenkiste for the L 5 GR, probably because we incubated the culture at
25.528.5°C as against 35°C , the temperature used by Van Steenkiste . Our results for the L 6-7 GR + OE are - after
18-21 days, probably as early as day 16 - better than Van Steenkiste's for the L 5 GR.
A working protocol for the growth of haemocytes cultures was developed. Primary cultures of haemocytes (+ fat body
fragments) from working bees larvae aged 4-5 and 6-7 days respectively were grown. Both the haemocytes (GR + OE)
and the cells of the fat body fragments (fat cells and oenocytes) were morphologically characterised and identified.
The GR + OF viability was evaluated for the time interval of 5-21 days of cells cultivation. Thus, the viability ranged between
81.1% (on day 5) and 903% (on day 9) with tile L 4-5 GR + OE during the 5-14 days period and from 90.4% (on day 14) to
60.7% (on day 21) with tile L 6-7 GR + OF during the 14-21 day interval.
Table 1 Viability values for the GR + OE cultures from L 4-5 and L 6-7
Time(days) VIABILITY(%)GR +OE (L4-5) GR+OE(L6-7)
5 81.1 ND
7 84.9 ND
9 90.2 ND
12 88.5 ND
14 88.5 90.4
18 ND 87.3
21 ND 60.7
ND= not determined
Figure 1 Comparative viability course for GR + OE (L 4-5 and L 6-7, 25.5-28.5°C, original) versus GR (L 5, 25° C, Van
Steenkiste, ) in primary cultures.
GR = Granulocytes
L 4-5 = 4-5 day old larvae
OE = Ocnocytoids
L 6-7 = 6-7 day old larvae
|ATTEMPTS TO IN VITRO CULTIVATE HONEY BEE (Apis mellifera L.)
I. Sorescu1, R. Tanasa1, Lenuta Gheorghe1, Aneta Mardare2, Gabriela Chioveanu2
1. Pasteur Institute, Bucharest
2. Diagnosis and Animal Health Institute, Bucharest
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