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Improved injection needles facilitate germline transformation of the buckeye butterfly Junonia coenia
 
Kahlia Beaudette1, Tia M. Hughes2,3, and Jeffrey M. Marcus1,2
1Department of Biological Sciences, University of Manitoba, Winnipeg, Canada
2Department of Biology, Western Kentucky University, Bowling Green, KY
3Center for Human Genetics Research, Vanderbilt University, Nashville, TN
BioTechniques, Vol. 56, No. 3, March 2014, pp. 142–144
Full Text (PDF)
Supplementary Material
Abstract

Germline transformation with transposon vectors is an important tool for insect genetics, but progress in developing transformation protocols for butterflies has been limited by high post-injection ova mortality. Here we present an improved glass injection needle design for injecting butterfly ova that increases survival in three Nymphalid butterfly species. Using the needles to genetically transform the common buckeye butterfly Junonia coenia, the hatch rate for injected Junonia ova was 21.7%, the transformation rate was 3%, and the overall experimental efficiency was 0.327%, a substantial improvement over previous results in other butterfly species. Improved needle design and a higher efficiency of transformation should permit the deployment of transposon-based genetic tools in a broad range of less fecund lepidopteran species.

Transgenic technologies greatly facilitate genetic research in insects. Drosophila researchers pioneered the use of transposon vectors to introduce genetic constructs into the germline (1). Transgenesis is now performed in many insect species (2), with transposon vectors engineered for insertional mutagenesis, mis-expression, creating mitotic clones, characterizing promoters, gene knockouts, and gene mapping (2-5). Implementing these versatile tools is an important step in developing genetic model systems (2).

The squinting bush brown (Bicyclus anynana) was the first butterfly to be transformed (6), but deployment of transgenic technologies in other butterflies has been slow. Our laboratory uses the common buckeye butterfly, Junonia coenia (Nymphalidae), as a model system to study the genetics, development, and evolution of color patterns (7, 8)(Figure 1). Transgenic tools would facilitate this work, so we began with handheld glass injection needles (Figure 1A) made from capillaries prepared identically to those used previously in Bicyclus(6, 9-11) (Supplementary Material). These needles, designated as Type 1 in this study, possess a long taper and small orifice (22 m) to minimize disruption of the ova cuticle.




Figure 1.  Injection needles and Nymphalid butterfly species used for injection needle comparisons and in germline transformation experiments. (A) Type 1 needles have a long taper and a small (22 m) orifice. (B) Type 2 needles have a steeper taper and a larger (67 m) orifice. (C) A Junonia coenia egg visualized via SEM microscopy. The micropyle is at the center of the image. (D) 5th instar larva Junonia feeding on the native larval host plant Agalinis tenuifolia. (E) Wildtype adult Junonia. (F) An adult Junonia derived from a G0 injected egg showing mosaic expression of enhanced yellow fluorescent protein (EYFP) from the pBac[3xP3 EYFP] transformation construct. Arrowhead indicates ommatidia expressing EYFP. (G) Larval F1 offspring of the mosaic individual in panel (F) showing expression of EYFP in each of its stemmata (larval photosensory organs). (H) The adult form of the same individual in (G) showing expression of EYFP in the ommatidia of the adult eye. (I) Adult Polygonia interrogationis. (J) Adult Vanessa atalanta. (Click to enlarge)


Unfortunately, our pilot injections produced 100% Junonia egg mortality (data not shown). Using identical needles in Bicyclus also produced a high mortality rate (often greater than 90% of the injected ova), poor survival to adulthood (typically less than 5%), and low overall efficiency (1 transgenic line per 1000 injections)(
6, 9-11). Thus, it was necessary to develop needles that could deliver genetic constructs while reducing ova mortality.

Optimization of injection needles was a critical innovation for germline transformation in Drosophila(12). The best Drosophila injection needles were determined empirically to have a steep taper and large orifice (67 m) (Figure 1B) (Supplementary Material). Survival to adulthood of Drosophila ova after injection with these needles, designated as Type 2, is 10%–50% (12), even though Drosophila ova are smaller than Bicyclus and Junonia ova (Figure 1C). Therefore, we investigated whether Drosophila-style needles could also reduce injected butterfly ova mortality.

Needles were filled with a 19:1 solution of TE buffer to Brilliant Blue FCF food dye (FD&C Blue 1, McCormick, Baltimore, Maryland). They were mounted onto a Picospritzer III microinjector (Parker Hannifin, Cleveland, Ohio) set at 20 psi and 0.004 s pulse duration for injections. Junonia ova were collected from host plants following one-hour oviposition bouts and aligned on thin strips of double-sided adhesive tape in plastic Petri dishes. Half of the ova were uninjected, while the remaining ova were injected with either Type 1 or Type 2 needles.

Method summary

Germline transformation is a powerful tool for the manipulation of insect genomes. We describe an improved needle design for butterfly ova injections that reduces post-injection mortality and demonstrate the use of these needles to efficiently transform the common buckeye butterfly, Junonia coenia.

We selected an ova injection site opposite the micropyle (Figure 1C), corresponding to the posterior of the butterfly embryo. Needles showing signs of wear, damage, or bluntness during injections were discarded immediately. After injection, ova were incubated in Parafilm sealed Petri dishes at 25°C and 70% relative humidity until hatching 4–6 days later. None of the 331 Junonia ova injected with Type 1 needles hatched, while 20 of the 349 ova (5.7%) injected with Type 2 needles survived (Table 1), suggesting that Type 2 needles produce less mortality in this species.

Table 1. 


Table 1.   (Click to enlarge)




To determine if needle performance was consistent, we tested both needle types in two additional species of Nymphalid butterflies, the question mark (Polygonia interrogationis) and the red admiral (Vanessa atalanta) (Figure 1I-J). Mated females were individually wild-caught with handheld nets, so ova from these species were limited. Therefore, small samples of ova were set aside as uninjected controls and for Type 1 needle injections, while the remaining ova were injected with Type 2 needles. The injections were performed as for Junonia. Type 1 needle injections produced lower survival to hatching rates than Type 2 injections in both Polygonia (0% versus 4.5%) and Vanessa (6.7% versus 13.3%)(Table 1). We suspect that the larger orifice of Type 2 needles injects fluid at lower velocity for a given pulse pressure and thus causes less internal disruption. Based on these findings, while Type 1 needles may sometimes be adequate, Type 2 needles improved hatch rates after injection in all species tested.

To confirm the efficacy of Type 2 needles for germline transformation, we injected 308 Junonia ova with piggyBac construct pBac[3xP3-EYFP](13) carrying the gene for enhanced yellow fluorescent protein (EYFP) under the control of an eyeless promoter. To maximize the likelihood of transformation, we manufactured and used new Type 2 needles on the same day. Ova were collected, injected, and incubated as previously described, except that eggs were injected with equal concentrations (500 ng/mL) of pBac[3xP3-EYFP] and helper plasmid pHsp82pBac containing the coding sequence of the piggyBac transposase driven by the Drosophila heat-shock promoter (13) in TE Buffer, mixed at a ratio of 19 mL plasmid solution to 1 L blue food dye. Approximately 21% of the injected embryos survived to hatching (Supplementary Material). Thirty-eight G0 animals survived to adulthood (3 showed mosaic expression of EYFP under fluorescent stereomicroscopy)(Figure 1F), and each was mated to 1–3 wildtype uninjected virgin individuals.

We col lected eggs from each family and screened larvae for EYFP in larval stemmata and adult eye. We identified one family (designated M9) with offspring expressing EYFP as both larvae and adults (Figure 1, Gand H). Family M9 EYFP-expressing individuals produced PCR products when amplified with EYFP-specific primers, while siblings lacking expression produced none (Supplementary Figure S1). Finally, we used inverse PCR to sequence flanking regions of the insertions. All M9 EYFP-expressing individuals shared a single transposon insertion site (Supplementary Figure S2), so we inbred this line, using fluorescence to select adults in each generation. We reconfirmed the presence of the EYFP gene by PCR after three generations.

We obtained a transformation efficiency of 3% for piggyBac in Junonia butterflies (Supplementary Table S1), comparable to transformation efficiencies obtained in Bicyclus(6, 9-11) and moths (14, 15). However, needle design modifications improved the overall experimental efficiency due to a dramatic increase in survival to hatching after injection (21.7% in Junonia versus 2.4%–14.2% in Bicyclus). Other measures of survivorship are similar in Bicyclus and Junonia, so improved needles alone facilitated this first report of successful Junonia transformation with only 1/10th the number of injections used in prior studies (Supplementary Material). Better needles make germline transformation technology practical in less fecund lepidopteran species because fewer ova are required. This should allow deployment of powerful genetic tools in many new species, potentially catalyzing advances in all fields for which lepidopterans are model organisms. Author contributions

J.M.M. conceived of the project and contributed to the design of the experiments, photography, data analysis, and manuscript writing. K.B. performed the injection needle comparison experiments, analyzed data, and co-wrote the manuscript. T.M.H. performed the Junonia transformation and verification experiments, analyzed data, and co-wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgments

We thank Jamie Roebuck, O. P. Perera, and Diane Ramos, for injection advice; Ernst Wimmer for plasmids; John Andersland, Tara Cox, Brooke Jackson, and Joey Simmons for assistance in the lab; and Roohollah Abbasi, Amber Gemmell, Ashley Haverstick, Bonnie McCullagh, Jacob Miller, Kristie Nybo, and three anonymous reviewers for helpful comments. Junonia butterflies were imported into Canada under Permit P-2009-04040 from the Canadian Food Inspection Agency. The National Science Foundation and the Commonwealth of Kentucky (EPS-0132295 and 0447479), the National Institutes of Health and the National Center for Research Resources (P20 RR16481), the Canadian National Science and Engineering Research Council (RGPIN386337-2011), and the Canada Foundation for Innovation (212382) supported this work. K. Beaudette was supported by a University of Manitoba Faculty of Science Summer Undergraduate Research Award and J. Marcus received support from the Canada Research Chair program (950-212382). This paper is subject to the NIH Public Access Policy.

Competing interests

The authors declare no competing interests.

Correspondence
Address correspondence to J. M. Marcus, Department of Biological Sciences, University of Manitoba, Winnipeg, Canada. E-mail: [email protected]


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