1968 - Wirth e Marston Mountage method

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May 1968] SCIENTIFIC NOTES 783
contrast microscope with a 100X oil-immersion objective
and a 10X ocular.
Preparations wore made permanent using a technique
similar to that of Smith (1943). The cover slip was
floated off in a solution of glacial acetic acid :absolute
alcohol .xylenc (1:1:1 , v/v) followed by immersion (1-2
min each) in 2 changes of absolute alcohol :xylene (1:1,
v/v). The final preparations were mounted in piccolyte.
Temporary mounts remained in good condition for 6
weeks. Staining for more than 10 min resulted in prep-
arations unsuitable for phase microscopy.
Breland's (1961) work with mosquitoes indicates that
rapid intermittent cell division occurs in insects. This
fact may explain why our best chromosome preparations
were obtained when the squashes were made about noon.
Difficulty in locating and preparing chromosomes of M.
rotundata for observation at other times indicated the
existence of cyclic cellular activity. The chromosome
number of the testes was 16 (Fig. 1), and in the ovaries it
was 32 (Fig. 2) (n and 2n, respectively). These figures
agree with numbers reported for Apoidca (Kcrr and
Laidlaw 1956) and further verify the haplo-diplo sex
inheritance observed in Hymenoptera (Sanderson and
Hall 1951, Whiting 1935).
REFERENCKS CITED
Breland, O. P. 1961. Studies on the chromosomes of
mosquitoes. Ann. Entomol. Soc. Amer. 54: 360-75.
Kerr, W. E., and H. H. Laidlaw, Jr. 1956. General
genetics of bees. Advances Genet. 8: 109-53.
Linkfield, R. L., P. B. Morgan, and C G. Haugh. 1967.
A new chromosome squash apparatus. Ann. Entomol.
Soc. Amer. 60: 706-7.
Sanderson, A. R., and D. W. Hall. 1951. Sex in the
honey-bee. Endeavour 10: 33-39.
Smith, S. G. 1943. Techniques for the study of insect
chromosomes. Can. Entomol. 75: 21-34.
"Whiting, P. W. 1935. Sex determination in bees and
wasps. J. Hercd. 26: 263-78.
A Method for Mounting Small Insects on
Microscope Slides in Canada Balsam1
WILLIS W. WIRTH2 AND NORMAN MARSTON8
Traditional methods for mounting insects in Canada
balsam have often proven unsatisfactory because of
shrinkage of delicate structures such as the antennae and
palpi in the highly viscous mounting medium. Clearing
in potassium hydroxide and subsequent gradual dehydra-
tion in a series of alcohols are very time consuming and
often require isolation of individual specimens, and sep-
arate treatment of the wings when adults arc being
mounted. While satisfactory mounts may easily be made
of relatively large and heavily sclerotized insects, mounts
of fragile insects are often inadequate for detailed taxon-
omic study. This problem has led many workers to try
other mounting media, but none has proven entirely
satisfactory.
Carter et al. (1920) and Lutz (1922) long ago called
attention to the advantages of dehydration and clearing
insects in carbolic acid (phenol). A method using phenol-
balsam for mounting specimens of Ceratopogonidae and
1 Accepted for publication December 14, 1967.
Chironomidae (Diptera) with little or no shrinkage has
been developed by the senior author (1961) and adapted
for mounting Cecidomyiidae by the junior author. The
specimens are placed in an alcoholic solution of phenol
for dehydrating, clearing, and relaxing which allows
them to be mounted in a phenol-balsam mixture. The
phenol then evaporates or polymerizes during the drying-
process, allowing a gradual transition into a permanent
balsam mixture. Additional major adavntages of this
method are that the chitin remains elastic and easy to
dissect, hairs and scales are not easily detached, wings
do not have to be treated separately, and as a result a
large number of specimens may be handled at 1 time or
held indefinitely in storage during the process.
SOLUTIONS REQUIRED
1. Liquified phenol, prepared by diluting pure phenol
(carbolic acid) crystals with enough absolute (100%)
ethyl alcohol to form a saturated solution, with a layer of
about y2 by volume of phenol crystals in the bottom of
the container. This solution may be prepared any time
before needed, but it turns slightly dark with age; only
fresh solution should be used in preparing the phenol-
balsam mixture.
2. A bottle of phenol-balsam mixture, prepared by mix-
ing equal parts (v:v) of liquified phenol and Canada
balsam of the consistency normally used for making
slides or a little thicker. This mixture must be pre-
pared at least a day in advance of use and no more than
3 weeks ahead, since it turns dark with age.
3. Canada balsam for replenishing the phenol-balsam
mixture in mounts as they arc dried in the oven.
4. (For Procedure 2 only.) Ten percent potassium hy-
droxide prepared by placing 1 part of a saturated solution
of potassium hydroxide and 9 parts (v:v) of distilled
water in a Stender dish or vial.
PROCEDURE 1. MOUNTING SPECIMENS COLLECTED IN ALCOHOL
Since insects collected in alcohol die with their append-
ages fully expanded and relatively relaxed, they may be
mounted directly into a phenol-balsam mixture after being
cleared and dehydrated in phenol. (Dry-collected or
heavily sclerotized specimens also may be mounted di-
rectly from phenol, although such mounts are generally
less satisfactory for detailed taxonomic study and may be
mounted according to Procedure 2.)
a. Place liquefied phenol in a Stender dish or vial and
transfer to it the specimens to be mounted. As many
specimens as desired may be placed in each dish or vial up
to % of the volume of the solution. The specimens should
be left in the solution for 1-24 hr depending on the length
of time necessary to relax and clear them. It may be
necessary to place unusually fragile specimens in a phenol -
70% alcohol mixture for about 5 min before placing them
in pure phenol to prevent shrinkage. Specimens may be
left in the phenol indefinitely; if the phenol crystallizes,
add 100% alcohol.
b. Place a drop of phenol-balsam mixture on a micro-
scope slide. Transfer the specimen from the phenol solu-
tion to the slide. Dissect and arrange the specimen on
the slide as required for study in each group of insects.
For females of Ceratopogonidae and Chironomidae, cut
off the head with dissecting needles and orient the face up-
ward. Cut off 1 wing and the abdomen, orienting the lat-
ter ventral side upward. For males, cut off 1 wing and
the head and orient as for the female; separate the ab-
domen just ahead of the genital segments and orient the
latter ventral side upward.
For Cecidomyiidae, remove both wings, the head, and
784 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA | Vol. 61, No. 3
the genitalia (if a male). Place the wings at the upper
right, the head at the upper left with the face upward, and
the body at the bottom with the legs inward. Male geni-
talia should be placed in the upper half of the slide with
either side upward.
A small fragment of broken cover slip should be placed
beside the thorax to prevent flattening and crushing the
specimen as the mixture contracts, through capillarity,
during drying. The wings or other structures may be flat-
tened under a separate cover slip if desired. Place only
1 specimen on each slide.
Place the cover slip on the specimen. Circular cover
slips of 18 mm diam are most satisfactory for Ceratopo-
gonidae and Chironomidae, 15 mm diam for Cecidomyi-
idae. Cover slips should be rested with 1 edge on the slide
to the side of the droplet and slowly lowered onto the
specimen, taking care that the small parts are not drifted
out of position. Drifting can be forestalled by adding
phenol-balsam mixture to that side of the cover slip with
a pipette. The mount should taper in thickness with the
head, wing, etc. on the thin side and the main body on
the thick side of the mount.
For delicate specimens it may be necessary to dilute
the phenol-balsam mixture with additional phenol. In
extreme cases the specimen may be mounted in pure
phenol to prevent shrinkage, in which case it may be
necessary to add additional 100% alcohol to the phenol to
prevent crystallization as the mount is prepared. Phenol-
balsam mixturemay then be added along the edge of the
cover slip and will gradually replace the phenol as the
slide dries.
c. Label the slide and place it in a drying oven at about
135°F for 1-2 weeks, or until the balsam has hardened
sufficiently to prevent slippage under moderate lateral
pressure. Replace the evaporated mixture with pure
balsam or phenol-balsam mixture every day or two until
no further evaporation takes place.
PROCEDURE 2. MOUNTING SPECIMENS COLLECTED DRY
In all groups of insects it is often desirable to mount
specimens which have been preserved dry or placed in
alcohol after desiccation. Specimens with a rigid exo-
skeleton may be mounted using the method just described,
but those species which collapse upon drying may require
treatment with potassium hydroxide to restore them to a
suitable condition. Potash treatment also may be nec-
essary to macerate and give better visibility to species
with opaque, thick, or dark-colored integument. Since
the muscles and other internal tissues are well preserved
(though cleared) in specimens mounted by Procedure 1,
it may be necessary to treat them also with potassium
hydroxide if the sclerotized internal structures are to be
studied or if the tissues interfere with phase-contrast
microscope examination.
a. Place the specimens in a solution of 10% potassium
hydroxide preheated to 200°F for 5-10 min. It is very
important not to leave winged specimens in the solution
too long, since the wings will lose their rigidity and will
wrinkle during the mounting procedure. Sometimes it
may even be necessary to dissect off the wings and
mount them separately before treating the specimen with
potash.
b. Transfer the specimens from the potassium hydroxide
solution to 70%—75% alcohol. In about 30 min the in-
ternal pressure of the specimens will cause the abdominal
segments, head, and appendages to expand beyond their
normal extended positions. This feature is desirable with
normally telescoped structures such as the female ovi-
positor.
c. Transfer the specimens to a solution of phenol and
70% alcohol for about 10 min and then into phenol-lOO^
alcohol solution. They should not be left in phenol for
more than 3 or 4 hr, since the phenol will cause the wings
to become adhesive, making mounting extremely difficult.
d. Arrange the specimen in a drop of pure phenol as
described under Procedure 1. Apply the cover slip, label,
and dry, using phenol-balsam mixture rather than balsam
to replenish the evaporating phenol.
REFERENCES CITED
Carter, H. F., A. Ingram, and J. W. S. Macfie. 1920.
Observations on the ceratopogonine midges of the
Gold Coast, with descriptions of new species. Part
I. Ann. Trop. Med. Parasitol. 14: 187-210.
Lutz, A. 1922. Contribution aux methods d'obserya-
tions microscopiques de biologiques. Ann. Biol.
Lacustre 11: 90-102.
Wirth, W. W. 1961. Instructions for preparing slides of
Ceratopogonidae and Chironomidae. Studia Entomol.
4: 553-4.
Regeneration of Appendages in Damselflies1 •-
DAVID E. PARVIN AND PAUL P. COOK, JR.*
Seattle University, Seattle, Washington
While rearing Zygoptera during ecological studies on
the Odonata, occasionally we observed that a naiad had
lost 1 or more legs and/or gills (caudal lamellae). When
this condition was noted it was recorded and notes were
kept on the extent of regeneration that occurred in sub-
sequent molts. No experimental studies were conducted.
The following observations refer to Ischnura pcrpari\i
Selys, /. ccrvula Selys, and Enallagma borcalc Selys, ex-
cept as noted.
Our information on regeneration of caudal lamellae is
adequate to state only that some increase in size occurs at.
each molt, and that the degree of regeneration in the first
molt following loss, or in subsequent molts, is variable.
Our data on regeneration of legs will allow the follow-
ing generalizations:
1. If an entire leg was lost:
a. In our observations it was always detached near
the base of the femur, rather than at the tro-
chanter as reported by Corbet (1962).
b. Regeneration resulted in a new leg with nearly
normal femur and tibia, although in some the
femur was the longer segment while in others the
tibia was the longer; the tarsus was 2 segmented,
the second tarsal segment was curved with a
curved spinelike apex, and the tarsal ciaws were
lacking (Fig. 1-3).
2. In /. perparva, if the tarsus is lost a new 2-seg-
mented tarsus is regenerated, with a nearly normal
apex, bearing a pair of tarsal claws which are
smaller than normal and lack the ventral subterm-
inal tooth (Fig. 4-7).
The size of a regenerated leg in the adult varied with
the stage in which the appendage was lost, but the degree
of regeneration at the first or subsequent molts following
1Insecta: Odonata.2 This research was supported by the Seattle University Ro-
search Committee, and some equipment used was purchased through
a grant from the Society of the Sigma Xi. Accepted for publica-
tion September 13, 1967.
3 Student and Assistant Professor, respectively, Department of
Biology.