Выращивание и инъекции<em> Manduca Sexta</em> Личинки для оценки бактериальной вирулентности
Summary
Метод, описанный здесь используется непосредственный впрыск энтомопатогенных бактерий в hemocoel из<em> Manduca Sexta</em> Личинками насекомых.<em> M. Sexta</em> Является коммерчески доступным и хорошо изученных насекомых. Таким образом, этот метод представляет собой простой подход к анализу хост-бактериальных взаимодействий с точки зрения одного или обоих партнеров.
Abstract
Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect’s immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.
Protocol
Representative Results
Discussion
Непосредственный впрыск М. Sexta личинок с энтомопатогенных бактерий, как описано здесь, служит простым и эффективным средством для анализа бактериальной вирулентности. Этот метод также легко адаптируется для различных испытуемых и / или условий. Бактерии могут быть получены разли?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить прошлом члены Goodrich-Блэр лаборатории: Саманта Orchard, Kimberly Cowles, Эрин Герберт-Tran, Грега Ричардса, Меган Менар, и Youngjin парка за их вклад в развитие этого протокола. Эта работа финансировалась Национальным научным фондом грантов IOS-0950873 и Национального института здоровья АЯРБ общение FAI084441Z.
Materials
| Reagent | Company | Catalogue number | Comments |
| 90 mm filter paper | Whatman | 1001 090 | |
| Glass filter holder | Millipore | XX1004700 | |
| Manduca sexta eggs | Carolina Biological Supply | 143880 | |
| Gypsy Moth Diet + agar | MP Biomedicals | 0296029301 | |
| 5.5 oz. plastic containers and lids | Solo Cup Company | URC55-0090 Pl4-0090 | |
| 1 oz. plastic containers and lids | DART Container Corporation | 100PC 100PCL25 | |
| 1x PBS | 137 mm NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4, pH 7.4 | ||
| Syringe | Hamilton | 80208 | 30 gauge, 0.375″ length, point style 2 |
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