Glycerol as a cryoprotectant agent to the entomopathogenic nematodes Heterorhabditis spp. and Steinernema spp.

Abstract

Glycerol as a cryoprotectant agent to the entomopathogenic nematodes Heterorhabditis spp. and Steinernema spp. Glicerol como agente crioprotetor dos nematoides entomopatogênicos AbstractThe difficulty of storage and conservation of entomopathogenic nematodes (EPNs) is one of the major obstacles for the expansion of its use in the control of biological pest and in the maintenance of collections of these organisms. The objective of this study was to evaluate cryopreservation as a storage and conservation method for EPNs, using glycerol as cryoprotectant. Infective juveniles (IJs) of the species Heterorhabditis amazonensis (RSC 05), H. bacteriophora (HP88), Steinernema feltiae (Sn) and S. carpocapse (IBCB-n02) were subjected to the following treatments: (A) immersion in glycerol at different concentrations (10, 13 and 15%); (B) different exposure times of the isolates to glycerol (24 and 48 hours); and (C) two freezing times in liquid nitrogen (LN) at -196 ºC (24 and 168 hours). Each treatment was replicated four times, and the design was completely randomized in a factorial 3x2x2 (glycerol concentrations x glycerol exposure time x freezing time in LN). IJs survival was evaluated after each exposure time to glycerol and freezing time in LN. Data were subjected to analysis of variance, and means were compared by the Tukey test. S. feltiae and S. carpocapse survived when exposed to glycerol at 10, 13 and 15% for 24 and 48 hours. After storage in LN for 24 and 168 hours, only S. feltiae survived when exposed to glycerol for 48 hours at concentrations of 10, 13 and 15%, with 40.5; 58.2; and 57.7% survival, respectively. S. feltiae was able to infect Galleria mellonella larvae after freezing. However, for the freezing time of 168 hours, 90% reduction in infectivity was observed. (24 e 168 horas). Cada tratamento teve quatro repetições e o delineamento foi inteiramente casualizado em esquema fatorial 3x2x2 (concentrações de glicerol x tempo de exposição ao glicerol x tempo de congelamento). A sobrevivência dos JIs foi avaliada após cada tempo de exposição ao glicerol e tempo de congelamento em NL, e os dados submetidos à análise de variância e as médias comparadas pelo teste de Tukey. S. feltiae e S. carpocapse sobreviveram quando expostos ao glicerol a 10, 13 e 15% por 24 e 48 horas. Após armazenamento em NL por 24 e 168 horas, somente S. feltiae sobreviveu quando exposto ao glicerol por 48 horas nas concentrações de 10, 13 e 15%, com 40,5; 58,2; 57,7% de sobrevivência respectivamente. S. feltiae foi capaz de infectar lagartas de Galleria mellonella após o congelamento, entretanto, para o tempo de 168 horas de congelamento foi observada uma redução de 90% na infectividade. Palavras-chave: Armazenamento. Controle biológico. Criopreservação. Nitrogênio líquido.