Rearing of larvae and pupae in the lab
In the lab, samples are poured into a large plastic container
(Fig. 11), and
the following steps go after:
a) Pupae are collected by using a micropipette and placed into
plastic jar of 35ml which are filled with approximately 25 ml
of no-chlorine water, the collecting number is written on the
lid.
b) Larvae in their last stage are separated to morph species and
placed into different small plastic jars with no-chlorine water
and small pieces of organic matter (Fig.
12), the collecting number is then written in the box.
c) For each morph species some larvae in their last stage are
separated for their preservation and slide-mounting. If these
larvae have much organic matter inside their guts, they have to
be placed into a solution of magnesium sulfate in order to purge
them. Larvae are preserved by putting them into hot water to kill
them and avoid them to become dark, then they are transfered into
jars with 75% alcohol for their later mounting.
d) For each box, larvae are separated in groups of 2 or 3 specimens
and put into jars of 35 ml with a little of organic matter.
e) Every day, containers with larvae are frequently checked looking
for pupae.
Water into rearing containers must be changed frequently to avoid
the development of pathogens. In the case that larvae remain for
a long time and some fungi are observed, it is recommended to
add a little bit of methyl blue to fight these fungi. In many
cases, the organic matter is gone after having the sample in the
lab for three days, then fish food (Tetra Min baby fish food)
is used to feed the larvae adding small portions every 3-4 days.(
Movie )
Once a pupa is found, this is transferred into a special container
to wait for the emerging of the adult. This special container
is a 5-ml vial where the pupa is placed and a 20-ml vial is put
over the first(Figs.
13 y 14).The
system allows that the adult emerges of the pupa and has space
for free movement without being in danger of drowning or damaging
the scams of its body. The corresponding larval exuviae is previously
put into a ¼ dram vial with 75% alcohol, and it is tied
together to the 20-ml vial with a rubber band. The pupa is also
placed into a container with clean water, and it is tied together
to the vial with the larval exuviae with a rubber band. The collecting
number is again written followed by a point and a rearing number.
The rearing number is consecutive corresponding to the number
of pupae associated with the larval exuviae found (LGCh 150.01;
LGCh 150.02; etc).
When the adult emerges, the pupal exuviae is put into a vial
together with its larval exuviae. Adults are placed in a dark
box for 24 hours to allow the hardening of the body and the 1800
rotation of the male genitalia to its normal position. After this
time, adults are stored into a freezer for their death and conservation
for later mounting.
The manipulation of larval and pupal exuviae must be done with
wet wood sticks to avoid the damage of setae and other structures
of the exuviae. Adults obtained from these pupae must have the
same number corresponding to those in the larval and pupal exuviae.
