J-ournal of the American Mosquito Control Association, 10(3):456_459, lgg4Copyrighr @ 1994 by the American Mosquito Coniiof Asiociation, Inc-

NEW TECHNIQUES FOR REARING BLACK FLIES FROMPUPAE (DIPTERA: SIMULIIDAE)

F. F. HUNTER,' S. G. BURGINI AND D. M. WOOD'

ABSTRACT. Simple techniques are described for coll:cting black fly pupae from streams using plasticstrips- and for rearing large numbers of adult black flies in inJxpensivJ J""ior"..r made of chicken wireand c-loth mesh netting, and 2 methods are described for rearing adult blact nies inaiviauarrv from p"pae.The first method of rearing individual black flies uses 1 . 5 -ml niicrocentrirujeiuuutes and the second useseasy-to-con_struct rearing chambers that provide moisture for the developin! pupa and water for the adultto imbibe. Instructions for assembly araprovided. Specimens obtained-u-s-iiithese rearing.tt"-G.r -eof museum quality.

Black fly larvae and pupae are found in a va_riety of streams. Species composition and pop-ulation densities can fluctuate widely throughoutthe season both within and among streams. Largeseries ofreared adults are often needed for tai-onomic purposes. For behavioral, physiological,or ecological studies, one might need large num_bers of flies of known age and history. Rearingadults from field-collected pupae is often the sim-plest way to obtain such material. However, itis time consuming to search streams by hand forlarge numbers of pupae.

Several authors have described ways in whichto rear black flies from eggs, early larval instars,or pupae through to adults, using elaborate andexpensive artificial streams (Bernardo et al. 19g6,see review in Edman and Simmons 1987). pub-lished methods for mass rearing adults from field-collected pupae have stressed the use of flow-through chambers or aquaria (Tarshis 1968). Briefaccounts also exist of rearing adults from indi-vidual pupae placed in tubes stoppered withmoistened cotton (Rubtsov 1956). Wood andDavies ( 1966) placed pupae singly in yz-in. (l .3-cm) square compartments of a sheet of plasticoverhead lighting grid, laid on top of wet filterpaper in a shallow tray. Each pupa was then cov-ered with a 2-in. (-5-cm) length of glass tubing,stoppered with cotton. Golini (1981) replacedthe lengths of glass tubing with a sheet of plastickitchen wrap. All of these previously suggesteddevices enclose the newly emerged adult in asmall space, and require that it be transferred toa holding chamber before pinning. Invariably theinner surfaces of the chambers become wet withcondensation, which tends to trap the adult, es-pecially small specimens.

The need for large numbers of healthy, reared

t Department of Biological Sciences, Brock Univer-sity, St. Catharines, Ontario L2S 3Al Canada.

2 Centre for I-and and Biological Resources Re-search, Agriculture Canada, Ottawa, Ontario Kl A 0C6Canada.

black fly adults for life history and taxonomicstudies led to the development of the followingsimple and inexpensive techniques that shouldprove useful to other simuliid researchers.

Collecting pupae: The method describedherein is suitable whether pupae are for mass orindividual rearing. It takes advantage ofthe factthat black fly larvae and pupae wilt readily col-onize artificial substrates such as plastic strips(Olejnicek 1980) that remain relativelv taut inthe current (Walsh et al. l98l).

Six 25 x lO-cm green garbage bag strips aretied at approximately l0 cm intervals to a lengthofplastic twine (Fig. lA). At least 30 cm of twineis left free at either end for attachment to sticksthat are pushed into the stream bed, thus securingthe strips so that water flows over them. De-pending on the type of stream, the series of stripscan be anchored transversely across the flow orin line with the flow. The latter method has prov-en especially effective in small shallow tricklesinhabited by some species of Greniera, Stegop-terna, Simulium (Eusimulium) spp., and otherheadwater specialists. It may be necessary to par-tially cover the strips with sand and other finebenthic matter if collecting Greniera and S/e-gopterna, which typically pupate in tiny cracksand crevices beneath the substrate.

Many larvae and pupae often are found on thestrips within 4-6 days. The strips are removedfrom the stream and transported back to the lab-oratory in plastic bags kept on ice. Strips arerinsed in a large basin of water to remove larvaeand other aquatic insects, thereby leaving onlythe black fly pupae (Fig. lB).

Mass rearing of adults: If species-pure sam-ples are needed, the plastic strips can be exam-ined under a dissecting microscope and pupae ofunwanted species removed with fine forceps. Thestrips are then ready to be placed in rearing en-closures. If mixed species assemblages are re-quired, strips are placed directly into enclosuresafter rinsing.

An inexpensive rearing enclosure can be con-structed by bending a l2O x 60-cm sheet of

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458 JourNlr or rrc Arrrpnrc.qr Moseurro Colrrnor Assocnrrolq Vor,. 10, No. 3

Fig. 3. Container for rearing individual flies of mu-seum quality. a. screw cap (loose, to avoid condensa-tion), b. simuliid pupa, c. sphagnum moss support forpupa, d. glass vial, e. shell vial, f. cotton (wet), g. poly-ethylene (Plastazoteo; foam disc, h. foam disc as seenfrom above, disc cut with sharp edge of plastic vial,i. support for shell vial, hole made with cork borer.

described beloq one for museum-quality spec-imens and one for checking species identity whenadult structures are needed for identification.

T ec h ni que for re arin g fl i e s i nd iv idual ly for r ap -id identification.' A simple, fast, and inexpensivemethod of obtaining reared adults is to cut aroundindividual pupae that are attached to the plasticstrips. Each pupa is then set on a moistened pieceof filter paper in a 1.5-ml Eppendorfo micro-centrifuge tubule. The lid of the tubule is thensnapped shut. All specimens from a single siteare stored together in a labeled Ziploco bag. Tu-bules are checked twice daily for emerged adults,which are visible through the opaque plastic.Adults are left to harden in the dark (to restrictactivity) for 24lr so that coloration patterns arewell defined.

The hardened fly can then be tapped to thebottom of the tubule, the lid opened, and 70o/oalcohol introduced from a squeeze bottle. Wehave found that specimens stored in this mannerare still usable even after 6 years, provided thatthe microcentrifuge tubules were tightly capped.For long-term storage, we recommend that thetubules be kept in an alcohol-filled sealer jar.

Unfortunately, coloration patterns tend to fadein alcohol-preserved specimens. Alternatively,therefore, tubules with reared flies can be placeddirectly into a freezer (-20"C) and the frozenflies mounted as described below.

Technique for rearing flies individually for mu-seum-quality specimens: The device describedhere (Fig. 3) overcomes many of the disadvan-tages of devices described by other authors. Itprovides a moist surface for the pupa, and forthe adult to drink from, in an otherwise dry en-vironment.

After removal from the streams, pupae are keptin petri dishes on wet sphagnum, cotton, or pa-per, until pharate adults appear ready to emerge.Then each pupa, either free, or still attached toa small piece of substrate, is placed among strandsof sphagnum moss protruding from a shell wialcontaining wet absorbent cotton inside a largerscrew-cap vial. Vials are labeled either by group-ing them in a larger labeled container, or insert-ing individual pre-prepared labels with the pu-pae. Each shell vial is "topped up" daily, withwater from a dropper, so that the amount ofmoisture surrounding the pupa remains rela-tively constant (the pupa in Fig. 3 is shown far-ther away from the top of the vial than usuallyplaced). Any water spilled inside the screw-capvial should be immediately removed with cot-ton. We have used both distilled water and waterfrom the same source as the pupae. Tap water isnot considered suitable because of added chlo-rine or similar antibacterial compounds, and per-haps because of excessive copper ions (as dis-cussed by Wood and Davies 1966).

To support the pupa we routinely use sphag-num moss, harvestedfrom living sphagnum mats,because it holds water better than cotton and ismore resistant to fungal attack. However, we havealso used absorbent cotton alone, changed be-tween each successive rearing. We have not triedcommercial peat moss, other types of mosses, orartifrcial sponges, although these could perhapsbe used if not contaminated with wetting agents.

The inner shell vial is filled with water. thecotton wick and sphagnum support inserted, andthe outside of the vial dried, before it is posi-tioned inside the larger screw-cap vial. The shellvial is held upright by a disc ofpolyethylene cutfrom a sheet offoam used as the pinning bottomfor unit trays @lastazote@). This disc is cut usingthe sharpened upper rim ofa plastic wial ofthesame diameter as the inside of the screw-cap vial.The small hole to support the shell vial, boredwith a cork-borer, is off-center to allow for betteraccess to the interior of the screw-cap vial withforceps or an aspirator.

Only pupae containing pharate adults close toemergence are placed in the screw-cap vials to

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minimize desiccation of pupal gills. The screwcaps are always kept loosely closed to preventmoisture from forming inside the screw-cap vi-als. Temperature shifts are avoided as much aspossible to avoid condensation. Vials are checkedtwice daily and those containing emerged adultsare set aside in the dark for 24h to harden anddarken. During this time adults often come tothe edge of the sphagnum to imbibe water. Mor-tality is higher during this period in the vials thathave dried up; some adults even force their wayinto the sphagnum in dried up vials, presumablyin search of water. We have not attempted toprovide a sugar meal, althoueh this might im-prove their longevity still further, provided theydo not become stuck in the sugar source.

After adults have remained 24 h in the dark,at room temperature, they are transferred into adeep freezer. All but the most hardy species arekilled within a few hours; some early spring spe-cies require longer. This method of killing re-quires no handling ofliving adults and, therefore,no opportunities of escape or damage duringhandling. The only disadvantage is that the wa-ter-filled inner shell vial will sometimes burst.

Flies frozen in their vials can be left for up toa week before being pinned. They still retain theirflexibility, but any disturbance should be avoid-ed to prevent damage to the frozen specimens.Each adult is attached by the right side of itsthorax to a small drop of shellac gel on a no. Ior 0 insect pin, I cm from the head ofthe pin(Shewell inMartin 1977). Pinned flies are im-mediately returned to the freezer for 4-6 months,or until completely freeze-dried. The associatedpupal exuviae and cocoon may either be storecin glycerol in a genitalia vial pinned beneath thefly, or glued when dry to the side of the pinbeneath the fly.

Between rearings, cotton and sphagnum areremoved from the shell vials and discarded. Thescrew-cap vials are wiped out to remove tracesof moisture and meconium and the shell vialsare cleaned, reloaded with wet cotton, their outersurfaces dried, and replaced in the screw-cap vi-

als. Between seasons, each component is thor-oughly washed and dried.

We thank the Ontario Ministry of Natural Re-sources for allowing this work to be done at theWildlife Research Station, Algonquin Park. Fig-

ure 3 was drawn by Ralph Idema. Funding wasprovided, in part, by Operating Grants from theNatural Sciences and Engineering Research

Council (Canada) to F. Hunter and M. Wood.

REFERENCES CITED

Bernardo, M. J., E. W. Cupp and A. E. Kiszeski. 1986.Rearing black flies in the laboratory: colonizationand life table statistics fot Simulium vittatum. Ann.Entomol. Soc. Am. 79:.610-621.

Edman, J. D. andK. R. Simmons. 1987. Maintainingblack flies in the laboratory, pp. 305-31 4. In: K. C.Kim and R.w. Merritt (eds.). Black flies: ecology,population management and annotated world list.Penn. State Univ. Press, University Park and Lon-don.

Golini, V. I. 1981. A simple technique for rearingpupae of Simuliidae and other Diptera. Entomol.Scand. 12:426428.

Martin, J. E. H. (editor). 1977. The insects and arach-nids of Canada. Part 1. Collecting, preparing, andpreserving insects, mites, and spiders. Agric. Can.Pub l .1643.

Olejnicek, J. 1980. Artificial substrates for quanti-tative ecological studies of preimaginal populationsofblackflies @iptera: Simuliidae). Acta Univ. Carol.Biol. 1977:372-375.

Rubtsov, I. A. 1956. Blackflies (Simuliidae). Faunaofthe USSR. Volume 6, Part 6. Academy ofSciencesof the USSR, Moscow and Leningrad. (Translatedfrom Russian in 1989 by Amerind Publishing Co.Pvt. Ltd., New Delhi, India.)

Tarshis, I. B. 1968. Use of fabrics in streams to collectblack fly larvae. Ann. Entomol. Soc. Am. 61:960-9 6 1 .

Walsh, D. J., D. Yeboah and M. H. Colbo. 1981. Aspherical sampling device for black fly larvae. Mosq.News 4l:18-21.

Wood, D. M. and D. M. Davies. 1966. Some meth-ods of rearing and collecting black flies @iptera:Simuliidae). Proc. Entomol. Soc. Ont. (1965) 96:81-90.