|Rajamohan, Arun - North Dakota State University|
|Rinehart, Joseph - Joe|
|Foster, Stephen - North Dakota State University|
|Leopold, Roger - Retired ARS Employee|
Submitted to: Journal of Economic Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/25/2012
Publication Date: 4/1/2013
Publication URL: http://handle.nal.usda.gov/10113/56905
Citation: Rajamohan, A., Rinehart, J.P., Foster, S.P., Leopold, R.A. 2013. Permeability barriers to embryo cryopreservation of Pectinophora gossypiella (Lepidoptera: Gelechiidae). Journal of Economic Entomology. 106(2):855-861.
Interpretive Summary: The pink boll worm, Pectinophora gossipiella is a major pest of the cotton crops. It is estimated that they cause more than USD 47 millions in damage per annum. Among the programs used to control the species are the use of transgenic crops and the sterile moth release. The research and development of control strategies have resulted in numerous mutant and transgenic strains of the moths that require enormous manpower and rearing cost. In addition there is also the risk of contamination and loss of strains that the researchers have to be mindful of. In recent years there has been a concerted effort to reduce the cost associated with rearing and securing the man-made and wild strains. The study being reported in this paper details a rough procedure to cryopreserve and store the embryos of pink boll worm in liquid nitrogen. The study shows that this lepidopteran species has a radically different egg shell morphology and possibly biochemical composition. These features dictate the difference in lepidopteran embryo cryopreservation procedure from the dipteran embryos procedure. A modified chemical treatment process was designed that permitted dehydration of the embryo without complete dissolution of the chorion. The study then proceeded to show that thus treated embryos are permit cryoprotectants to permeate into the embryos. This was confirmed by the changes in the supercooling point of the embryos that were exposed to ethylene glycol. An average of 7% of the embryos hatched from the cryopreserved embryos. 76% of the hatched larvae pupated and 59% of the pupae eclosed. This understanding of the embryonic membrane/shell morphology and their permeability characteristics gained in this study will form the basis for the future fine tuning of the procedure and expansion of this protocol to other lepidopterans of economic importance as well as those in fragile ecosystems.
Technical Abstract: The aim of this study was to develop a method to cryopreserve the embryos of the pink bollworm moth, Pectinophora gossypiella (Saunders). Previously developed dipteran cryopreservation protocols were not directly adaptable to use with the embryos of this lepidopteran species. Physiochemical and electron microscope observations revealed substantial differences in the structure of the chorion, wax layer and vitelline membrane complex when comparing the cryopreservable embryonic stages of P. gossypiella and dipteran embryos. Thus, the initial steps dealing with dechorionation and permeabilization were ineffective and had to be altered. Exposure to the sodium hypochlorite-based chorion removal step decreased P. gossypiella embryo viability to a very low level. Survival increased and permeability was evident when an alkane wash was used as the first step in the procedure. Following the alkane treatment with a surfactant yielded the maximum exchange of cryoprotectant (CPA) with water as evidenced by a significant lowering of the supercooling point of the CPA-loaded embryos. The remainder of the cryopreservation and storage recovery protocol for P. gossypiella was similar to those developed for dipteran embryos. Survival of recovered, hatched embryos to adulthood was approximately 7%, from which a reproducing colony of these moths was started.