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. 2019 Dec 20;9(24):e3456.
doi: 10.21769/BioProtoc.3456.

Preparation of Red Palm Weevil Rhynchophorus Ferrugineus (Olivier) (Coleoptera: Dryophthoridae) Germ-free Larvae for Host-gut Microbes Interaction Studies

Abrar Muhammad  1   2 , Prosper Habineza  1   2 , Youming Hou  1   2 , Zhanghong Shi  1   2
Affiliations

Preparation of Red Palm Weevil Rhynchophorus Ferrugineus (Olivier) (Coleoptera: Dryophthoridae) Germ-free Larvae for Host-gut Microbes Interaction Studies

Abrar Muhammad et al. Bio Protoc. .

Abstract

Red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, is a devastating pest of palm trees worldwide. RPW gut is colonized by diverse bacterial species which profoundly influence host development and nutritional metabolism. However, the molecular mechanisms behind the interactions between RPW and its gut microbiota remain mostly unknown. Antibiotics are usually employed to remove gut bacteria to investigate the impact of gut bacteria on insect fitness. However, administration of antibiotics cannot thoroughly remove gut bacteria for most insect species. Therefore, establishing germfree (GF) organisms is a powerful way to reveal the mutual interactions between gut bacteria and their insect hosts. Here, we describe a protocol to generate and maintain RPW GF larvae, being completely devoid of gut bacteria in laboratory. RPW GF larvae were established from the dechorionated fresh eggs which were reared on the sterilized artificial food under axenic conditions. The establishment of GF larvae set a solid foundation to deeply elucidate the molecular mechanisms behind the interactions between RPW and its gut microbiota.

Keywords: Germfree larvae; Gut microbiota; Host physiology; Insect symbiosis; Rhynchophorus ferrugineus.

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Conflict of interest statement

Competing interestsThe authors declare that they do not have any competing interest.

Figures

Figure 1.
Figure 1.. The four different life stages of red palm weevil, Rhynchophorus ferrugineus
Figure 2.
Figure 2.. Non-dechorionated and dechorionated eggs of red palm weevil, Rhynchophorus ferrugineus.
(A) Non-dechorionated eggs are only washed with autoclaved distilled water only and (B) dechorionated eggs were washed with 10% sodium hypochlorite solution (NaClO), 75% ethanol and sterilized distilled water.
Figure 3.
Figure 3.. The gut from the fifth RPW larvae
Figure 4.
Figure 4.. Culture-dependent verification of bacterial growth in the guts of RPW larvae from different treatment cohorts.
No bacterial colony is observed in the larvae guts from germ-free (GF) group: dechorionated eggs reared on antibiotic treated food (Dechorionated + food with antibiotic). However, bacterial colonies were found in other three treatment cohorts: DNA (dechorionated eggs + food without antibiotics), NDA (non-dechorionated eggs + food with antibiotics) and CR conventionally reared (non-dechorionated eggs + food without antibiotics).
Figure 5.
Figure 5.. Verification of gut bacteria-derived PCR products with bacterial 16S rRNA-based PCR assays.
GF (Dechorionated eggs + food with antibiotics), DNA (dechorionated eggs + food without antibiotics) NDA (non-dechorionated eggs + food with antibiotics) and CR conventionally reared (non-dechorionated eggs + food without antibiotics).
Figure 6.
Figure 6.. Quantification of bacterial colony forming units (CFUs) in the guts of RPW larvae from different treatment groups.
The number of CFUs of each plate was counted 24 h after incubation at 37 °C. A One-way ANOVA (Tukey’s HSD post hoc multiple comparisons) was run to detect the statistical differences of bacterial CFU across the treatments. The letters indicate significance at P < 0.05. GF (Dechorionated eggs + food with antibiotics), DNA (dechorionated eggs + food without antibiotics), NDA (non-dechorionated eggs + food with antibiotics) and CR conventionally reared (non-dechorionated eggs + food without antibiotics).
See this image and copyright information in PMC

References

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