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PUBLISHED DATE: - 03-12-2024
DOI: -
https://doi.org/10.37547/tajabe/Volume06Issue12-03
PAGE NO.: - 12-17
TECHNOLOGY FOR IN VITRO REARING OF
PARASITICS
Khabiba Khidoyatova
Researcher, Vice Dean - Tashkent State University of Economics, Uzbekistan
Rasul Jumaev
Professor, Doctor of Agricultural Sciences, Tashkent State Agrarian University, Uzbekistan
INTRODUCTION
As the main procedure in biological control of
agricultural insect, mass production of several
species of parasitoids has been conducted in
Uzbekistan. Their factitious hosts for mass
production are still the eggs or larvae of insects. For
instance, the eggs of Helicoverpa armigera and
Agrotis Segetum are used for mass rearing
Trichogramma spp. The larvae of Helicoverpa
armigera is the factitious host for mass producing
Bracon habetor Say or Bracon greeni. However,
searching for more convenient and cheaper
methods of parasitoid’ mass rearing is still an
important challenge in biological control.
Manufacture of the simulated “host eggs” and
“larvae” for in vitro rearing these parasitoids is
developed rapidly too. In this paper, artificial diets
and simulated hosts of these parasitoids are
described[1;2;6;7].
METHODS
1) Artificial diets
: For Trichogrammatidae.
Best 3 species Trichogramma were tested: T.
pintoe, T. evanescens, T. chilonis. The first 2 species
are indigenous in Uzbekistan, the others were
introduced from China[1;2;3;4].
This Trichogramma were collected from Tashkent
province, Buka district, and reared in the
laboratory on Galleria mellonella.
a) Pupa hemolymph of Galleria mellonella (heated
RESEARCH ARTICLE
Open Access
Abstract
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in a water bath at 60 0C for 4 or 5 min) 45.5+1.5 %,
chicken yolk 22.5+1,0.3 %, milk 10% / 20.5+0.5 %,
inorganic salts
11,5+1,0.3 % (Neisenheimer’s
mixture salt NaCl7.5 g, KCl0.1 g, CaCl20.2 G,
NaHCO30.2 g, H2O 100 ml), Penicillin 400/ml.
Streptomycin 400 units/ml.
b) Pupa hemolymph of Helicoverpa armigera
42+1,0.2 %, chicken yolk 25+1,0.8 %, milk 10% /
22.5+2,0.2 %, inorganic salts 10.5 +0.5 %,
Penicillin 400/ml. Streptomycin 400 units/ml.(Fig-
1;2).
c) Pupa hemolymph of Agretus segetum 43+2,0.2
%, chicken yolk 24+1,0.8 %, milk 10% / 21.5+0.6
%, inorganic salts 11.5+1.0 %, Penicillin 400/ml.
Streptomycin 400 units/ml. The development of T.
chilonis, reared in vitro successively for many
generation, is showed in Table 1[1;2;3].
2) Artificial diets for Braconidae
: Best 2 species
Braconinae were tested: Bracon hebetor Say,
Bracon greeni. This Bracon were collected from
Tashkent province, Buka district, and reared in the
laboratory on Galleria mellonella and Helicoverpa
armigera[4;5;6;7].
Table 1.
DEVELOPMENT OF HYMENOPTERA, REARED IN VITRO
SUCCESSIVELY FOR 10 GENERATION
Generation
1-5
6
7
8
9
10
% parasitism
100
100
100
100
100
100
% adult emergence
89+3
88+3
87+4
86+4
86+3
85+3
% adult with expanded
wings
82+3
82+2
81+3
81+2
80+3
80+2
% pupa development
93+3
92+4
92+3
90+4
90+2
87+3
% normal adult
86+2
86+3
86+4
85+2
82+2
80+3
♂: ♀ genders proportion
2:8
2:8
2.7
2.6
2:5
2:4
Preparing of artificial mediums.
1.
Insect hemolymph collection: Pupa were
immersed in water bath at 600C for 6 or 7 min to
avoid blackening of the hemolymphy. After surface
sterilization with alcohol and need sterile
condition.
2.
Chicken embryo extract collection: The
Chicken embryo extract, only it should be the egg
yolk and need sterile condition.
3.
Milk: Fresh cow milk or 10% powdered milk
solution also need sterile condition.
4.
Inorganic mixture salt: Use Neisenheimer’s
mixture salt (NaCl7.5 g, KCl0.1 g, CaCl20.2 G,
NaHCO30.2 g, H2O 100 ml).
It is defined there is albumen, oil, water when it is
checked ingredients of master caterpi
llar’s type
that belong to Bracon in nature. Besides, Heliothis
armigera Hb, Agrotis segetum Sciss and Galleria
mellonella’s pupa liquid is used for research (Fig
-
2).
Natural milk 10% mixture is added to hemolymph
and hicken yolk is added diet medium and put at
ultraviolet lamp. It is rotated for 5 minutes at 2000
second speed in centrifuge. Diet medium should be
kept in the clean, without microbe room and at
20°C cool [1.3.5;6;7].
After medium has been ready, it is placed in special
artificial caterpillars which are cleaned with 75%
ethyl spirit made of politilen.
MANUFACTURE OF SIMULATED "ARTIFICIAL
EGG-CARDS AND LARVAE"
1) Artificial “egg
-
cards”
: There are 2 types of
artificial “egg
-
cards”[1;2;3].
a) Tri-
ring “egg
-
cards” 2 pieces of plastic film are
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used. The semispherical concaves are made on the
upper plastic film. Artificial medium is poured into
concaves fully (but without overflow) with a micro-
syringe or micro-pipette. The bottom plastic film
has no concaves. The upper and bottom plastic
films are separated and stretched tightly by three
plastic ring with different inner diameters, in our
Cass they are: 5:5 cm, 5.4 cm, 5.2 cm respectively.
b) Bag-
form “egg
-
cards”: One half of a piece of film
is fulled of concaves with medium and is covered
by another half of film. Three sides of film are
sealed. Convex side of capsules are exposed and
between the concave side and bottom half of the
same piece of film there should be space for
aeration. The size of plastic film depends upon the
number of semispherical concaves made on
film[1;2;3].
Fig-1. Bag-form artificial “egg - card” and egg.
2) Produce depicted artificial larvae
: A small
part (1x1) of parafilm is extended 2x4 cm,
absorbed in 75% alcohol for 15 minutes. Then it is
dried with sterile printed paper, folded as a sack
and fixed in order to stick both sides.0,5 ml of
mediums is placed by pipette into each parafilm
box.
Fig-2
.
а)
Аrtificial larvae and pupa (
Trichogramma chilonis
Ishii)
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When parafilm box is fulled with mediums, it yields
such depicted maggot.15-20 small holes are
opened with sterile entomologic needle for
properly prepared artificial caterpillars. All the
processes of the preparation are required to carry
out in sterile room [1;3;4;5;7].
RESULTS OF IN VITRO REARED PARASITOIDS
It was showed in the Table 1 that the pupae
hemolymphy of either G.melonnellan or
H.armigera Hb could be used as the main
component of the artificial diet for the
development of Trichogrammatidae. There was no
significant difference in their parasitism, survival,
percentage of pupation, adult emergence and
reproductively when the pupae hemolymphyof
G.melonnella was used instead of that of
H.armigera Hb.
It is obvious in the research, prepared all mediums
of diets are harmed with Trichogrammatidae
generation and put there eggs. But it is observed
dying because of inconvenience medium of diets
for developing parasite generation. According to
diet mediums component development of
Trichogrammatidae parasite was rather prolonged
when G.melonnella hemolymphy and inorganic
salt’s quantity was more in diets medium. In fact
quantity of protein and oil is more in the structure
of hemolymphy as well as it is considered
convenient
for
development
of
parasite
maggot[1;2;3].
Fig-3
.
Artificial larvae (
Bracon hebetor
Say)
Thus, from above nominated artificial diet
mediums, pupaе hemolymph of G.melonnella
45.5+1.5 %, chicken yolk 22.5+1,0.3 %, milk 10% /
20.5+0.5 %, inorganic salts 11,5+1,0.3 % composed
medium of diets is defined as a suitable for normal
nourishment
and
development
of
Trichogrammatidae generation in order to rear
trichogramma parasite[1;2].
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Fig-4. In vitro reared Braconidae.
2) We chose two type of Bracon
. Bracons, Bracon
hebetor Say and Bracon greeni, this type is tolerant,
durable in extreme condition. It is practiced by
means of choosing convenient state in developing
each type to be harmed diet mediums with Bracon
and in this condition it is put in thermostat(Fig-4).
Damaging the type of Bracon with diet mediums at
30±1°C temperature, at 68±3% moisture.
Thus, in order to develop and consume of
Braconidae generation well from above mentioned
artificial diet, mediums hemolymph wax moths
(G.melonella) 40,04%, chicken yolk 30,03%, milk
10% 29,03% is confirmed as the best diet medium
to rear Braconidae parasite[1;5;6;7].
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