Effect of cryopreservation on the pre-hatching behavior in the Mexican fruit fly Anastrepha ludens Loew (Diptera, Tephritidae)

Authors: Rajamohan, Arun; Rinehart, Joseph - Joe; LEOPOLD, ROGER - Retired ARS Employee

Submitted to: Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2017
Publication Date: 2/1/2018
Citation: Rajamohan, A., Rinehart, J.P., Leopold, R.A. 2018. Effect of cryopreservation on the pre-hatching behavior in the Mexican fruit fly Anastrepha ludens Loew (Diptera, Tephritidae). Cryobiology. 80:38-42. https://doi.org/10.1016/j.cryobiol.2017.12.007.

Interpretive Summary: Mexican fruit fly (Anastrepha ludens) embryos exhibit an unusual pole reversal behavior prior to hatching. The embryos developing within the egg case turn around completely and move their anterior end to the narrower tailend of the egg. Thereafter they proceeed to gnaw through the shell and hatch out. This behavior is seemingly energy exhaustive and therefore in this study it was used as an index to determin if pole reversal behavior is afflicted significantly when the embryos are permeabilized and cryopreserved. We found that a significant number of the embryos did not complete the pole reversal behavior after cryopreservation. Further analysis indicated that cryopreservation only partially contributed to this debility and a significant portion of the problem was due to the chemical and physical pretreatments (dechorionation, permeabilization and cryoprotectant loading).

Technical Abstract: In a sampling of untreated embryos of Mexican fruit fly, Anastrepha ludens, the cumulative hatch percentage was 84.77±7.8% of which ~70% of the larvae eclosed through the posterior pole of the egg. This is due to an unusual and seemingly energy demanding act of flipping of the fully developed pre-hatch larva from the wider anterior to the narrower posterior pole of the egg. In this study, significantly lower proportion of cryopreserved larvae were observed to exhibit pole flipping behavior. Therefore we decided to investigate the effects of cryopreservation (by vitrification) and the pretreatments involved therein on this behavior. Pretreatments followed by vitrification caused a delay in hatching, reflecting slower development by ~10 hours. This resulted in a large number of the treated embryos either eclosing from the anterior end of the egg or not eclose at all although they appeared to be fully developed and active. Among the untreated controls 24.0% of the embryos eclosed from the anterior pole. After permeabilization and cryopreservation 83.20% and 54.52% (adjusted hatch) of the embryos were noted to hatch this way. However our analysis of the hatch count after the treatments show that factors contributing to the embryos’ inability to properly invert polarity is not solely due to cryopreservation but also due to the pretreatment procedures including dechorionation and permeabilization. In fact, the permeabilization pretreatment contributed the highest to this phenomenon.