Molecular Characterization and Functional Analysis of the Hb-hsp 90-1 Gene in Relation to Temperature Changes in Heterorhabditis bacteriophora

Abstract

Understanding how entomopathogenic nematodes respond to temperature changes and have adapted to the local environment is crucial to improve their potential as biocontrol agents. In order to improve understanding of Heterorhabditis bacteriophora's potential adaptability to future climate changes, full-length cDNA and the corresponding gene of heat shock protein 90 (Hsp90) were isolated and fully characterized. The reproductive potential of the Apulian strain of H. bacteriophora increased when the temperature rose from 23 to 30°C, but no reproduction was found at 12°C. Expression analyses revealed that Hb-hsp90-1 was differentially expressed in Infective Juveniles (IJs) and adults (hermaphrodites, females and males). Up-regulation of Hb-hsp90-1 was higher during the recovery process in Galleria mellonella larvae than adults, thus confirming the protective role of Hb-hsp90-1 in coping with the host environment. Silencing of Hb-hsp90-1 resulted in a significant reduction (76%) in the expression level. Silenced IJs took longer than untreated nematodes to infect G. mellonella, showing that Hb-hsp90-1 could be also involved in chemosensation. Furthermore, the number of adults and IJs recovered from G. mellonella infected with silenced nematodes and incubated at 30°C was higher than that obtained from G. mellonella infected with untreated nematodes. These data confirm the crucial role of Hb-hsp90-1 allowing acclimation to increased temperatures and modulation of the recovery process.

Keywords: EPN; acclimation; gene duplication; heat shock protein; silencing; thermotolerance.

FIGURE 1

Phylogenetic analysis of the deduced amino acid sequences of Hsp90 isoforms from Heterorhabditis bacteriophora reveals the presence of distinct groupings and the duplication of the retropseudogene Hsp90-2 occurred relatively recently in Heterorhabditis bacteriophora.

FIGURE 2

Deduced amino acid sequence of the Hb-Hsp90-1 of Heterorhabditis bacteriophora. The five Hsp90 signature sequences are shaded in light gray. GxxGxG motif is indicated in bold. The consensus leucine zipper sequence is double underlined. The consensus LxxLL sequence is indicated in bold underlined. The cytoplasmic Hsp90 sequence motif (MEEVD) is underlined and shaded in dark gray.

FIGURE 3

Comparison of intron-exon structure of Hb-hsp90-1 of Heterorhabditis bacteriophora compared with those of Meloidogyne artiellia, Brugia pahangi, and Caenorhabditis elegans. Exons are shown as boxes and introns as V-shaped lines. The amino acid at intron junctions indicated conserved positions between nematodes.

FIGURE 4

Expression of the Hb-hsp90-1 in Infective Juveniles (IJs) and adults. Bars indicate standard error of mean data (n = 3). (**P < 0.01).

FIGURE 5

Expression profiles of the Hb-hsp90-1 of Heterorhabditis bacteriophora at 12°C, 23°C, and 30°C in IJs (A) and adults (B) in aqueous solution. Bars indicate standard error of mean data (n = 3). Significant differences (*P < 0.01; **P < 0.05) were found between control and treated nematodes.

FIGURE 6

Expression profiles of the Hb-hsp90-1 of Heterorhabditis bacteriophora at 30°C in Infective Juveniles (IJ), (A) and adults (B) during parasitic development. Bars indicate standard error of mean data (n = 3). Significant differences (**P < 0.05) were found between temperature control and higher temperature.

FIGURE 7

Expression of Hb-hsp90-1 in Infective Juveniles (IJs) of Heterorhabditis bacteriophora treated with dsRNA. Control consisted of untreated IJs for 24 h at 23°C. Significant differences (**P < 0.01) were found between treated and untreated IJs.

FIGURE 8

Light microscopy observations of RNAi mediated phenotypes of Infective Juveniles (IJs) of Heterorhabditis bacteriophora treated with dsRNA Hb-hsp90-1 after 24 and 48 h soaking at 23°C.

FIGURE 9

Mean percentage mortality of Galleria mellonella larvae after 24 and 48 h following exposure to dsRNA treated Infective Juveniles (IJs) and untreated Infective Juveniles at 23 and 30°C (A). Different letters above bars indicate statistical differences. Untreated and silenced Infective Juveniles (IJs) were transferred on Petri dishes, containing water agar layer, in presence of Galleria mellonella larvae to assess nematode attraction (B).

FIGURE 10

Number of adults and/or Infective Juveniles recovered from Galleria mellonella larvae after 5 and 10 days from infection with treated and untreated Infective Juveniles (IJs) at 23 and 30°C. Bars with the same letter have no significant differences (P < 0.05).

FIGURE 11

Effects of temperatures on the reproductive potential of treated and untreated Heterorhabditis bacteriophora inside Galleria mellonella larvae after 10 days infection. (A) untreated nematodes at 23°C; (B) dsRNA treated nematodes at 23°C; (C) untreated nematodes at 30°C; (D) dsRNA treated nematodes at 30°C.