Temperature-Dependent Galleria mellonella Mortality as a Result of Yersinia entomophaga Infection

Abstract

The bacterium Yersinia entomophaga is pathogenic to a range of insect species, with death typically occurring within 2 to 5 days of ingestion. Per os challenge of larvae of the greater wax moth (Galleria mellonella) confirmed that Y. entomophaga was virulent when fed to larvae held at 25°C but was avirulent when fed to larvae maintained at 37°C. At 25°C, a dose of ϳ4 ؋ 10 7 CFU per larva of a Y. entomophaga toxin complex (Yen-TC) deletion derivative, the Y. entomophaga ⌬TC variant, resulted in 27% mortality. This low level of activity was restored to near-wild-type levels by augmentation of the diet with a sublethal dose of purified Yen-TC. Intrahemocoelic injection of ϳ3 Y. entomophaga or Y. entomophaga ⌬TC cells per larva gave a 4-day median lethal dose, with similar levels of mortality observed at both 25 and 37°C. Following intrahemocoelic injection of a Yen-TC YenA1 green fluorescent protein fusion strain into larvae maintained at 25°C, the bacteria did not fluoresce until the population density reached 2 ؋ 10 7 CFU ml ؊1 of hemolymph. The observed cells also took an irregular form. When the larvae were maintained at 37°C, the cells were small and the observed fluorescence was sporadic and weak, being more consistent at a population density of ϳ3 ؋ 10 9 CFU ml ؊1 of hemolymph. These findings provide further understanding of the pathobiology of Y. entomophaga in insects, showing that the bacterium gains direct access to the hemocoelic cavity, from where it rapidly multiplies to cause disease. The Gram-negative bacterium Yersinia entomophaga was isolated from the cadaver of a Costelytra zealandica (Coleoptera: Scarabaeidae) larva, and has proven consistently pathogenic by per os challenge to this host, as well as to a wide range of coleopteran, lepidopteran, and orthopteran species (1). A 6-day median lethal dose (LD 50 ) was determined as 2.9 ϫ 10 4 Y. entomophaga cells per C. zealandica larva. Following per os challenge with a bacterial dose of ϳ1 ϫ 10 7 Y. entomophaga cells per larva, the larvae typically vomited, expelled frass pellets, changed color from a healthy gray to amber and then a moribund brown, and exhibited reduced feeding activity. By 48 h postingestion, Y. entomophaga cells could be visualized within the hemocoelic cavity of the larvae, after which time death ensued (2).The main virulence determinant of Y. entomophaga is an insect-active toxin complex (TC) derivative termed "Yen-TC" (3). TCs were first identified in the genome of Photorhabdus luminescens (4) and have since been identified in other bacterial genera, including members of the genus Yersinia (5). Typically, TCs are comprised of three proteins, TC-A, TC-B, and TC-C, which combine to form the insect-active complex (6). Yen-TC is comprised of seven subunit proteins: two TC-A-like proteins (YenA1 and YenA2), a TC-B-like protein (YenB), two TC-C-like proteins (YenC1 and YenC2), and two chitinases (Chi1 and Chi2), which combine to form the insect-active TC. Recent structural analysis has revealed that both the Yen-TC and the P. lumine...

Serratia proteamaculans Strain AGR96X Encodes an Antifeeding Prophage (Tailocin) with Activity against Grass Grub (Costelytra giveni) and Manuka Beetle (Pyronota Species) Larvae

“…PO activity was assessed by collecting hemolymph from the larvae by cutting off a leg and collecting 15 l of outflowing liquid (hemolymph). This liquid was immediately transferred to a prechilled microcentrifuge tube and processed as previously described (52). When required, 10-l aliquots of hemolymph were assessed for bacterial content by using serial dilutions, as described above, and assessed by light microscopy using an Olympus BX50 light microscope and an Olympus DP-72 digital camera.…”

The Draft Genome Sequence of the Yersinia entomophaga Entomopathogenic Type Strain MH96T

“…Y. entomophaga is consistently pathogenic by per os challenge to this host, as well as a wide range of lepidopteran, coleopteran, and orthopteran species, with death of the insect typically occurring within 2–5 days of ingestion [ 11 , 12 ] . Aside from hemocytes, there are no obvious sites of Y. entomophaga colonization in the insect [ 12 , 13 ]. At 37 °C, aggregates of Y. entomophaga were observed both in the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) larval hemocoel and in vitro in Luria-Bertani broth.…”

“…At 37 °C, aggregates of Y. entomophaga were observed both in the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) larval hemocoel and in vitro in Luria-Bertani broth. Electron microscopy of these in vitro cell aggregates revealed the presence of polar fimbriae [ 13 ].…”

“…Rhs proteins are composed of a conserved N -terminal region and a divergent C -terminal “tip” region of 100–200 amino acids [ 16 ] that can encode the toxin effector component [ 17 , 18 , 19 ]. The Yen-TC causes loss of gut epithelial integrity, allowing the bacterium to gain entry to the insect hemocoelic cavity [ 13 , 20 ].…”

Caracterização e avaliação do papel do gene <i>wcbE</i> de <i>Burkholderia seminalis</i> linhagem TC3.4.2R3 na interação microbiana.

“…The Yen-TC toxin mediates the entrance of the bacterium into the hemocellic cavity of the insect, through the dissolution of the midgut of the larvae. Once inside the hemocellic cavity, the bacterium multiplies rapidly and suppresses the immune system of the host, in addition to possibly limiting the growth of the endemic microbial community (HURST et al, 2015). Thus, it is possible to note the importance of temperature in the regulation of the gene expression, controlling biological activities, virulence and strategies of survival in microorganisms.…”