Short Technical Reports dence to Catharine A. Conley, Department of Cell Biology, MB24, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA, 92037, USA. Internet: cassie@ scripps.edu Received 13 March 1995; accepted 30 August 1996.
Electromechanical Microinjection of Pink Bollworm Pectinophora gossypiella Embryos Increases Survival BioTechniques 22:496-499 (March 1997)
Catharine A. Conley and Maureen R. Hanson1 The Scripps Research Institute La Jolla, CA 1Cornell University Ithaca, NY, USA
ABSTRACT We report and compare methods and apparatus for injection of pink bollworm, Pectinophora gossypiella (Saunders), embryos. Injection with an electromechanical device resulted in 59% survival of embryos. Previous techniques relying on a mechanical manipulator resulted in 8% survival. Pink bollworm (PBW) embryo injection technology is based in part on methods developed for injection and genetic transformation of Caenorhabditis elegans ([Maupas] Dougherty) and Drosophila melanogaster (Meigen) with substantial alterations in both method and apparatus to accommodate special conditions of PBW biology, behavior and morphology. The microinjection methodology described here has direct application to other difficult-to-inject insects and invertebrates.
INTRODUCTION Analysis of the genetics and molecular biology of Drosophila melanogaster (Meigen) has benefited tremendously from experiments based on germ-line genetic transformation (9). Modern genetic analysis and manipulation of other insect species would benefit similarly from methods for genetic transformation of target insects (3,5,6). Towards this end, we are developing transformation protocols for pink bollworm, Pectinophora gossypiella (Saunders). A method, such as microinjection, for introducing transforming constructs into early embryos is one technical obstacle to be overcome (10). Other investigators injecting Lepidopteran eggs have reported low (30%) survivability (10). Our results demonstrate that electromechanical injection is a great improvement over simple mechanical injection.
MATERIALS AND METHODS Collection of Eggs Pink bollworm (PBW) adults were kept in 1-gallon, screen-covered containers in a humidified incubator on a 12 h:12 h light:dark cycle. Females were allowed to oviposit onto glass microscope slides placed atop the holding container at the beginning of the egglaying period. Egg-covered slides were removed approximately 45 min to 1 h after placement. Eggs not to be injected were removed with a razor blade. The region of the slide with eggs to be injected was ringed with embedding wax to form a small reservoir approximately 1-mm deep that was filled with water-saturated light mineral oil (E.R. Squibb & Sons, Princeton, NJ, USA). Oil improved the microscope image, prevented the eggs from drying excessively and kept the DNA solution from drying in the needle bore and clogging the tip. Needles One-millimeter-diameter omega capillaries (World Precision Instruments [WPI], New Haven, CT, USA) were pulled with a Sachs Flaming Micropipette Puller PC-84 (Sutter Instrument, San Rafael, CA, USA) to create a stout-necked needle shape with a 2–5-µm bore. Capillaries were backfilled with nucleic acid solution and mounted in a ferrule injection arm connected to a WPI picopump. DNA solution was delivered from the needle by a 400-ms pulse at 4.133 × 10-3 N/m2 pressure. DNA Injection Needles were back-filled with a sterile solution of potassium phosphate/ KCl buffer (10 mM KH2PO4, pH 7.0, 50 mM KCl) containing 300 ng/µL DNA to be injected. The needle was manipulated either by a manual micromanipulator (Model M-3; Narishige Medical Systems, Greenvale, NY, USA) or an electromechanical micromanipulator (Model 5171; Eppendorf, Madison, WI, USA). The manual micromanipulator was bolted to a 2-cm-diameter by 30-cmVol. 22, No. 3 (1997)
Table 1. Comparison of Survivorship of Embryos Injected with a Manual Micromanipulator and an Electromechanical Micromanipulator
Number of Embryos % Survival Manual Electromechanical Manual Electromechanical Injected Transferred to medium Total developed
10 101 2292
880 642
852
401
100 24 8.4
100 73 45
Table 2. Survivorship of Embryos Injected with an Electromechanical Manipulator and Washed with 2% Triton X-100 Before Transfer to Medium
Number of Embryos Injected
273
% Survival 100
Transferred to medium
207
76
Total developed
162
59
long, solid-steel cylinder anchored to a 1.5-cm × 30-cm × 30-cm aluminum plate resting on size 9 neoprene rubber stoppers to dampen vibration. We used a preprogrammed “impale” function for injections with the electromechanical micromanipulator (2). After piercing the embryo, the picopump was triggered to deliver a pulse of nucleic acid-containing buffer. Injections were observed at 10× magnification through a Zeiss-Reichart compound interference contrast microscope (McBain Instruments, Tustin, CA, USA) modified to provide a large working distance between the objective and stage. The microscope was mounted on the same vibration-dampened, aluminum base plate as was the manipulator. The camera port of the microscope was fitted with a SONY® DXC107A camera and CMA-D2 camera adapter (McBain Instruments) that allowed videotape recording of the injection process. We replaced the standard, fixed microscope stage with a 360° rotating stage to allow us to rotate randomly oriented embryos for posterior injections. Either a manual or a custommade, computer-controlled XY slide manipulator was attached to the top of the rotating stage. The computer-controlled slide manipulator was driven by two stepper-motors integrated with the EggHunt ( 1995, David Ferguson, University of California, Riverside, Chemistry Dept.) PC computer program. The EggHunt program scanned Vol. 22, No. 3 (1997)
all of the eggs to be injected (by means of the camera port) and recorded each egg’s XY position. After an injection, clicking the “next egg” button in the program caused the next egg to be brought into the field of view. Larval Rearing After completion of the injections, the wax wall was removed, and the slide was held on its side to drain off the oil. The injected embryos were kept at 20°–25°C in a covered dish for 6 days and inspected daily. Embryos close to eclosion were removed from the glass slide with a camel’s hair paint brush and placed into medium (1) in 100-mL plastic rearing chambers, twenty embryos per chamber. Some embryos were washed to complete oil removal with a solution of 2% Triton® X-100 (Sigma Chemical, St. Louis, MO, USA) just before transfer. Larval rearing chambers were kept at 28°C in constant darkness in a humidified incubator. Insects were allowed to develop to at least the ultimate larval instar. Insects were sacrificed as larvae, pupae or adults, and nucleic acids were extracted for use in a separate study. Some adults were mated for G1 egg collection. RESULTS Injections with the electromechanical micromanipulator resulted in more BioTechniques 497
Short Technical Reports than a fivefold increase in survivorship over those done with the manual manipulator (Table 1). Embryos injected with the manual manipulator usually died before eclosion (Table 1). However, with the micromanipulator system, the majority of injected eggs completed embryonic development and were transferred to a rearing diet. Many developed embryos failed to eclose completely and thus died trapped within the eggshell. Since this may have been due to the mineral oil used during injection, some developed embryos were washed with 2% Triton X-100 just before transfer to the medium. Survivorship among electromechanically injected eggs that were washed with detergent increased to 59% (Table 2).
The sex ratio of injected pink bollworms was approximately 50:50 male: female (data not shown). Survivability among sham-injected controls (phosphate buffer/KCl alone) did not differ significantly from those injected with DNA solution (data not shown). DISCUSSION Comparison of survival between embryos injected with the manual manipulator with survival rates of those injected with the electromechanical device demonstrates a greater than fivefold increase in survival with the electromechanical device. The insects we study oviposit a substantial proportion of nondeveloping eggs, often as many as 20%–25%. Thus, the improved survival rate we observed may be close to the maximum possible. The electromechanical manipulator was designed for microinjection of mammalian oocytes and embryos. It is capable of precise and repeatable movement with no “drift” of the apparatus during use, unlike the manual micromanipulator. Perhaps more importantly, the injection needle held by the electromechanical device is isolated from tremors introduced by the operator’s hands, as control is effected electronically from a remotely placed joystick. This remote operation also lessens the possibility of repetitive stress injury to the operator. The chorion of insect eggs is difficult to pierce (7). In Drosophila spp. the chorion is removed before injection (8), but this is not practical in all species; pink bollworm mortality increases greatly following dechorionation. Piercing the PBW chorion with the manual manipulator is tedious and difficult. In contrast, the speed of the electromechanical stepper motors causes the needle to quickly pierce the leathery chorion of the pink bollworm egg without substantial distortion or disruption of the embryo. Most importantly, the precision and accuracy obtainable with the electromechanical device results in minimal damage to the embryo and eggshell upon withdrawal of the injection needle, since the path of exit is identical to the path of entry. Because the manual manipulator is manu-
ally driven, hand tremors are unavoidable. These small tremors are amplified at the needle’s tip and often result in substantial damage to the injected egg (Table 1). With the pink bollworm, significant mortality after embryos are transferred to the rearing medium is not uncommon. Approximately 6% of the uninjected embryos develop to the point of eclosion but die within the eggshell (data not shown). It is our observation that the mineral oil used during injection is an additional source of mortality because eggs retain an oil coating even after transfer to the medium. We saw eclosing larvae struggling to escape the viscous oil, which may occlude their mouthparts as they cut through the
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eggshell. Oil-fouled larvae are unable to clean their mouthparts of oil and become lethargic, do not feed and eventually die. A detergent wash of eggs just before eclosion resulted in a 14% increase in survival compared to eggs that were not washed. Since genetic transformations can be rare, increasing the survival of injected embryos increases the chance of obtaining transgenic bollworms. Our improvement in the survival of injected embryos will assist in the production of transgenic PBW for use in genetic control programs (4). In addition, since embryo injection using an electromechanical micromanipulator is much easier to learn and safer to the operator than the mechanical manipulator, a larger cross section of laboratory workers can perform injections, further increasing productivity. The EggHunt computer program automates the movement of eggs attached to microscope slides into the field of view during injections. Since the XY position of the slide holder, and thus the position of the eggs on the slide, is recorded by EggHunt, many eggs can be moved into the field of view, videotaped and returned to exactly the same position at a later time. This allows for time-lapse photography studies of embryonic development. More information on the EggHunt program is available from the authors. Although our described microinjection system was specifically designed to introduce nucleic acid-containing buffer solutions into pink bollworm embryos, it should be usable with minor modifications to introduce a variety of substances—e.g., endosymbionts, proteins, hormones and toxicants—into a wide range of insect and other invertebrate embryos with little mortality from injection trauma. Because of its increased targeting accuracy and low trauma induction, this method also may be usable for maternal injection studies. REFERENCES 1.Adkisson, P.L., E.S. Vandersant, D.L. Bull and W.E. Allison. 1960. A wheat germ medium for rearing the pink bollworm. J. Econ. Ent. 53:759-762. 2.Anonymous. 1994. Operating Manual for Eppendorf, Micromanipulator 5171. Eppendorf Vol. 22, No. 3 (1997)
GmbH, Hamburg, Germany. 3.Blackman, R.K. and W.M. Gelbart. 1989. The transposable element hobo of Drosophila melanogaster, p. 523-529. In D.E. Berg and M. Howe (Eds.), Mobile DNA. American Society for Microbiology, Washington, DC. 4.Fryxell, K.J. and T.A. Miller. 1995. Autocidal biological control: a general strategy for insect control based on genetic transformation with a highly conserved gene. J. Econ. Ent. 88:1221-1232. 5.Handler, A.M. 1993. Technology for transforming the germ line in economically important insects: current status and prospects for advancements, p. 79-91. In Management of Insect Pests: Nuclear and Related Molecular and Genetic Techniques. Proceedings of an international symposium on management of insect pests: nuclear and related molecular and genetic techniques. International Atomic Energy Agency and the Food and Agriculture Organization of the United Nations. 19-23 October 1992. 6.Handler, A.M., S.P. Gomez and D.A. O’Brochta. 1993. A functional analysis of the P-element gene-transfer vector in insects. Arch. Insect Biochem. Physiol. 22:373-384. 7.Miller, L.H., R.K. Sakai, P. Romans, R.W. Gwadz, P. Kantoff and H.G. Coon. 1987. Stable integration and expression of a bacterial gene in the mosquito Anopheles gambiae. Science 237:779-781. 8.Roberts, D.B. (Ed.) 1986. Drosophila: A Practical Approach. IRL Press, Oxford. 9.Rubin, G.M. and A.C. Spradling. 1983. Vectors for P-element-mediated gene transfer in Drosophila. Nucleic Acids Res. 11:63416351. 10.Tamura, T., T. Kanda, S. Takiya, K. Okano and H. Maekawa. 1990. Transient expression of chimeric CAT genes injected into early embyros of the domesticated silkworm Bombyx mori. Jpn. J. Genet. 65:401-410.
This work was supported by a grant from the California Cotton Pest Control Board (93-0614). The mention of proprietary products does not imply endorsement. Address correspondance to John J. Peloquin, Department of Entomology, University of California, Riverside CA 92521, USA. Internet:
[email protected] Received 9 May 1996; accepted 22 August 1996.
John J. Peloquin, Stephen T. Thibault, Leo P. Schouest, Jr. and Thomas A. Miller University of California, Riverside Riverside, CA, USA
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