Parasite Rearing and Specimen Preparation

Identification of parasitic Hymenoptera is based on characters found in the adult stage.
For most Hymenoptera, especially those parasitic on aleyrodids, species identification
is based on characters of the adult female.

Rearing Methods
I utilize two methods for rearing parasites; there are advantages and disadvantages to each one.
A) Bulk Rearing: Whitefly infested leaves are placed in a paper can (or other sealable container)
     for about two weeks. Parasite specimens are extracted from the leaf material and either
     stored in 70-95% ethanol or placed directly in a clearing solution. The advantages of using
     this method are that it is fast, easy and usually yields more specimens with little effort. The
     disadvantages of using this method are that the host-parasite association may be uncertain
     since there may be more than one host species present on the leaf. Also, predators that may
     eat the host and/or the parasites are not screened as well. It is common to rear egg and
     leafminer parasites in these samples, which are fairly easy to distinguish from whitefly
     parasites; however, there may be more than one species of whitefly present on the leaf
     which makes associating the parasite with the correct host uncertain.
B) Isolation: The advantages of using this method are that there is no uncertainty as to host-parasite
     association and predators are screened so they can not eat the host or parasite.
     Cut a small leaf disk containing a single whitefly pupa, particularly those suspected of
     being parasitized from the leaf, place it in a small vial, seal the vial with a small ball of
     cotton, and hold it for about 2 weeks. Emergence may be enhanced by placing the vials in a
     humidity chamber. A make-shift humidity chamber can be made by placing a salt solution at the
     bottom of a jar, construct a platform over the salt solution, place the vials on a platform and close the jar.

Specimen Preparation
All parasite specimens can be stored in 70% ethanol. If they are to be used for DNA
studies, then it is better to deep-freeze them or store them in 95-100% ethanol and maintain them in a deep-freezer.
Species identification of whitefly parasites, particularly for Encarsia and Eretmocerus species,
often requires that specimens be slide mounted and observed with the compound microscope.
Generally, eulophid and platygastrid parasites are better observed in alcohol or dry on points or card mounts.

Slide Mounting
I utilize two medium for slide mounting; there are advantages and disadvantages to each one.
A) Modified Berlese Medium (Hoyers): The advantages of using hoyers are that it is very fast,
     specimens do not lose as much of their color in the clearing process and the structural
     characters are more easily seen. The disadvantage of using this method is that it is not a
     permanent medium. The life span of this medium is somewhat unpredictable. Specimens
     mounted in hoyers may last from a few months to over 30 years before the medium
     crystallizes and the specimen is no longer observable. I mount most of the specimens
     collected from surveys in hoyers, and mount type specimens and other important
     specimens that need to be archived for long periods of time in Canada Balsam.

1. Clear several specimens by placing them in Nesbitt’s solution overnight (less sclerotized
     species may require 4-5 hours). I use small watchglasses for soaking specimens.
2. Place a small drop of hoyers in the center of the microscope slide.
3. Transfer the specimen to the drop of hoyers. The specimen should be facing downward
     (dorsum up) with the head facing towards you.
4. Extend the wings, legs and antennae
5. Place the coverslip on top of the specimen quickly and tapping on the top of the coverslip to
     lock the specimen into place.
6. Press gently on the coverslip until the medium reaching the coverslip edge.
7. Label the slide with the appropriate data.
8. Place the slide in a drying oven or on a slide warmer for several weeks for the hoyers to dry.
9. Seal the coverslip by ringing the coverslip with GLPT Electronic Varnish so that the humidity
     does not contact the hoyers; this prolongs the life of the slide mount.

B) Canada Balsam - The advantage of using Canada Balsam is that it is a permanent medium.
    Holotype specimens should be mounted in balsam. If I have a series of specimens, I will mount
    most of the paratypes in Canada Balsam and a few in hoyers. The disadvantages of using Canada
    Balsam are that the process takes longer, specimens lose more of their color and the structural
    characters are not as easily observed. Antennae sometimes will collapse, body setae are more
    easily lost and the surface sculpture is not as clear.

1. Place specimens in cold 10% KOH overnight (at least 4-5 hours) to clear the body contents.
2. Transfer the specimens to water to remove excess KOH.
3. Place specimens in 70% ethanol for 5 minutes (Do not stain parasite specimens or use isopropyl alcohol).
4. Place specimens in 95% ethanol, if antennal collapse occurs, then try bringing the specimens
    through intermediate concentrations of ethanol (70% to 80% to 90% ethanol).
5. Transfer specimens to a mixture of half clove oil and half 95% ethanol for several minutes.
6. Transfer specimens to100% clove for about 10 minutes.
7. Place a small drop of Canada Balsam in the center of the microscope slide, dilute with a drop of
     xylene, if necessary.
8. Place the specimen face down (dorsal side up) in the balsam with its head facing towards you.
9. Extend the wings, antennae and legs
10. Place the coverslip on top of the specimen. Apply gentle pressure to the top of the coverslip
     until the balsam reaches the coverslip edge.
11. Label the slide and place the slide in a drying oven or on a slide warmer until dry.

                       Aphelinidae                                USA: Florida
                           Encarsia                                    Gainesville
                           nigricephala                               24 VII 2002
                           Dozier                                         G. A. Evans    
                                                                              Bemisia tabaci on
                          #2002-340                                  Hibiscus
                                Hoyers                                     rosa-sinensis