Virulence of Serratia Strains against Costelytra zealandica

Abstract

Strains of Serratia spp. showed a high level of virulence when injected into the hemocoel of larvae Costelytra zealandica, with Serratia entomophila, S. plymuthica, and S. marcescens showing significantly higher virulence than S. proteamaculans. Toxicity was independent of the amber disease-causing plasmid pADAP, suggesting a generalized Serratia toxin.

The genus Serratia (Enterobacteriaceae) contains insect pathogenic strains (5) which are usually considered to be opportunistic or facultative pathogens, as they are often avirulent to insects when present in the digestive tract but are lethal upon entering insect hemocoel following injury or stress (3). In contrast, strains of Serratia entomophila and S. proteamaculans containing the pADAP plasmid consistently produce amber disease after ingestion by the New Zealand grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae) (7, 9). Amber disease is a gut-colonizing disease caused by a cluster of three genes termed sep (S. entomophila pathogenicity) genes on the pADAP plasmid, with the bacteria invading the hemocoel only after a long period of chronic infection (4, 7, 10). The sep proteins show significant similarity to the toxin complexes produced by bacterial entomopathogens Photorhabdus luminescens and Xenorhabdus nematophila, which are usually nematode vectored, leading to death of the nematode by septicemia, but can also be orally active (1, 13, 14). Thus, while oral activity of amber disease-causing strains has been well characterized (9, 10), the effects of coelomic delivery were not known. In this study, we examined virulence and pathology of pADAP-bearing and pADAP-free Serratia strains against C. zealandica larvae, estimated the intracoelomic lethal doses of the bacteria, and examined the resultant pathology.

The bacterial strains bioassayed in this study are listed in Table 1. Bacteria were produced in an overnight shake culture in Luria-Bertani (LB) broth at 37°C for Escherichia coli and 30°C for Serratia (7). Cell densities of bacteria were estimated from CFU after dilution plating with phosphate-buffered saline (PBS) onto LB agar plates.

TABLE 1.

Estimated LD₅₀s for intracoelomic delivery of Serratia strains with and without the pADAP plasmid and E. coli against C. zealandica larvae

Treatmenta	Presence of pADAP plasmidb	Median injected dose (cells/larva)	Mortality (%) at day 6c	Overall LD50 (cells/larva) (95% confidence interval)d
Serratia spp.
S. proteamaculans 2746	+	1,282	52	880 (52-15,019)
S. proteamaculans 142	+	1,313	56	802 (59-10,941)
S. plymuthica 590	−	1,202	83	22 (3-114)
S. marcescens 363	−	1,355	96	<5
S. entomophila 154+	+	1,253	89	<5
S. entomophila 154−	+	1,247	92	<5
Controls
PBS			6
E. coli DH10B	−	914	14

^aBacterial strains held in the AgResearch Insect Pathogen Culture Collection.

^b+, with plasmid; −, without plasmid.

^cMortality was averaged over all five dose rates.

^dCalculated using the three log LD₅₀s for each strain (with 2 df). For S. plymuthica 590 Replicate 1, the LD₅₀ was 30 cells larva⁻¹ (the minimum detectable level), so a value of 30 was used in the calculation.

pADAP-bearing strains S. entomophila 154+, S. proteamaculans 2742, and S. proteamaculans 142 and pADAP-free strains S. plymuthica 590, S. marcescens 363, and E. coli DH10B (11) were injected into healthy grass grub larvae. Two microliters of diluted suspension of bacterial culture was injected into the hemolymph through the larval head capsule by using a 1-ml Hamilton syringe with a 30.5-gauge needle (8) to achieve doses ranging from 10⁰ to 10⁶ cells per insect. Larvae for uninfected controls were injected with equal volumes of PBS. The symptoms of toxicity were recorded and the mortalities monitored over time postinjection. The experiment was repeated on three occasions in a completely randomized design. The experimental unit was a group of eight larvae. Inoculated larvae were individually placed in wells of 24-well tissue culture plates, held in the dark in an incubator at 15°C, and observed until 14 days postinjection. Data were analyzed using Genstat 7.1 software (Genstat UK) with treatment mortalities corrected for control mortalities and median lethal doses estimated by probit analysis.

To assess the ability of bacteria to survive and grow within the insect hemocoel, we compared the S. entomophila A1MO2(pADK6) E. coli DH10B and labeled by green fluorescent protein using plasmid pGFPuvsm (6). Bacteria were delivered as described above, with doses of approximately 10¹ to 10⁵ cells larva⁻¹ for E. coli DH10B and approximately 10¹ to 10³ cells larva⁻¹ for the S. entomophila strain containing pADK6. Larvae were bled up to 48 h postinjection when 20 μl of hemolymph was removed from each larva and dilution plated with PBS onto LB agar plates containing the appropriate antibiotics (6). Resultant CFU were assessed by visualization of green fluorescent protein-based fluorescence after exposure on a UV transilluminator. The experiment was carried out with six larvae for each dose rate of each strain, and three larvae assessed at each time point for all dose rates of each strain. Pathology was monitored by examination of hemolymph extracts by phase-contrast microscopy and examination of thin sections of embedded tissue fixed from moribund larvae.

All Serratia strains caused mortality after injection into the larvae. Larvae inoculated with doses of ≥10⁴ cells larva⁻¹ of Serratia strains began to die within 24 h postinjection, while larvae inoculated with doses of ≤10³ cells larva⁻¹ tended to die after 48 h postinjection. After six days, S. entomophila 154+ and 154−, S. plymuthica 590, and S. marcescens 363 produced the highest mortality, with estimated 50% lethal doses (LD₅₀s) that were significantly (P<0.05) lower than those of S. proteamaculans 2746 and 142, which were in turn significantly (P<0.05) lower than those of controls, including E. coli DH10B (Table 1). Disease symptoms included lethargy and a darkening of the larvae, but gut clearance, a primary symptom of amber disease, was not a symptom here. Following intracoelomic inoculation of the pathogenic S. entomophila strain containing pADK6, the bacteria grew rapidly within the hemolymph, with the CFUs reaching 10⁹ cells per ml of hemolymph in 48 h. In contrast, after injection of E. coli DH10B, no CFUs were recovered, indicating that cells were rapidly cleared from the hemolymph shortly after inoculation. Microscopic visualization of hemolymph revealed strong bacterial growth accompanied by vacuolization of the hemocytes, leading to cell lysis. Infection was accompanied by a severe disruption of the midgut epithelium in moribund larvae.

These experiments show that the Serratia strains tested had a high virulence against larvae of the New Zealand grass grub, C. zealandica, when injected directly into the hemocoel and can be considered potential pathogens by the definition of Bucher (2). The effects of Serratia sp. inoculation were clearly distinct from those produced by E. coli DH10B, which did not cause mortality even when applied at a concentration of 10⁵ cells larva⁻¹. Serratia-induced mortality could be produced with inoculation of very low numbers of cells, and in this regard, our results coincide with those of Lysenko (12) for intracoelomic inoculation of S. marcescens into caterpillars. Microscopic examination of larvae through the disease process indicates that Serratia virulence is related to the ability of the bacteria to avoid or overcome the insect host's defenses and proliferate in the hemocoel producing cytotoxic effects against both hemocytes and gut epithelial tissue.

This rapid pathology accompanied by cytotoxic effects is clearly distinct from the chronic amber disease produced by strains of S. entomophila and S. proteamaculans (9, 10). No relationship was found between the presence of the amber disease-causing plasmid pADAP and virulence in these experiments. Indeed, the matched strains S. entomophila 154+ and 154− showed similar virulences, while the plasmid-bearing S. proteamaculans strains showed the lowest virulence of the Serratia strains tested. Furthermore, the characteristic amber disease symptoms of cessation of feeding and gut clearance (9, 10) were not observed in these experiments. Thus, the pathogenicities of these strains of Serratia to grass grub larvae following coelomic injection appear to be related to a generalized Serratia toxin rather than caused by the toxin complex/Sep-type gene complex found on the pADAP plasmid.