Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa

The time course of clearance of an injected dose of 10⁶ CFU ml^?1 hemolymph of Pseudomonas aeruginosa ATCC 9027 in larvae of the tobacco hornworm, Manduca sexta, has been examined in detail. The clearance process has been subdivided into three stages during which the rates of reduction in concentration of circulating viable bacteria were clearly different. Contributions of hemocyte reactions to bacterial clearance were examined during stages I and II. During stage I (0–2 hr postinoculation (PI), nodule formation produced a dramatic reduction in circulating bacteria by entrapping over 90% of the injected dose in the first 30 min. Phagocytosis of bacteria by circulating hemocytes and subsequent intracellular digestion contributed significantly to reductions in circulating bacteria during stage II (2–8 hr PI). Viable cells of the virulent P. aeruginosa P11-1 were trapped in nodules as efficiently as the less virulent 9027 during the first 30 min after injection into M. sexta. Bacteria of strain P11-1 were also phagocytosed by hemocytes during stage II, however, phagocytosed bacteria were observed less frequently in P11-1-treated insects and intracellular digestion of these bacteria was only rarely observed. The increased virulence of P11-1 in larvae of M. sexta may be due to less efficient phagocytosis by circulating hemocytes and to insensitivity of this strain to killing reactions in nodules and following phagocytosis.     

Bombyx mori prohemocyte division and differentiation in individual microcultures

We followed the fate of microcultured Bombyx mori prohemocytes in vitro. Prohemocytes isolated from larval hemolymph (day 1 of 4th instar) were maintained for 4-11 days in serum-free MGM-450 medium and some of them underwent mitotic division. Over 60% of the non-dividing prohemocytes differentiated to plasmatocytes or granulocytes. Some of the granulocytes subsequently transformed to spherulocytes. Of the dividing prohemocytes, 59.2% of the daughter cells differentiated into other types of hemocytes such as plasmatocytes, granulocytes and spherulocytes, and the remainder divided into new prohemocytes. Four of these renewed prohemocytes generated daughter cells composed of plasmatocytes and granulocytes. These results suggest that prohemocytes possess the properties of stem cells, and that plasmatocytes and spherulocytes may be terminally differentiated cells, whereas granulocytes, at least in part, may be a transient form of spherulocyte. Oenocytoids were not produced, suggesting that the lineage of oenocytoids differs from that of other types of hemocytes and that it is determined before release from hemopoietic organs.     

Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis

Originally from tropical Asia, the Red Palm Weevil (RPW Rhynchophorus ferrugineus (Olivier)) is the most dangerous and deadly pest of many palm trees, and there have been reports of its recent detection in France, Greece and Italy. At present, emphasis is on the development of integrated pest management based on biological control rather than on chemical insecticides, however the success of both systems is often insufficient. In this regard, RPW appears to be one pest that is very difficult to control. Thus investigations into the natural defences of this curculionid are advisable. RPW hemocytes, the main immunocompetent cells in the insect, are described for the first time. We identified five hemocyte cell types from the hemolymph of R. ferrugineus: plasmatocytes (∼50%), granulocytes (∼35%), prohemocytes (∼8%), oenocytes (∼4%) and spherulocytes (∼3%). SEM observations were also carried out. Some aspects of RPW interaction with non-self organisms, such as Saccharomyces cerevisiae and the entomopathogen bacterium, Bacillus thuringiensis (Bt), are discussed. Plasmatocytes and granulocytes were involved in nodules and capsule formation as well as in the phagocytosis of yeast. The hemocyte response of RPW larvae to sub-lethal doses of commercial products containing Bt was examined. In vivo assays were carried out and Bt in vegetative form was found in the hemolymph. After a diet containing Bt, the number of total hemocytes, mainly plasmatocytes, in the RPW larva hemolymph declined sharply (∼12%) and then remained at a low level, while the number of other circulating cells was almost unchanged.     

亚洲玉米螟幼虫血细胞的包囊行为

根据光镜和电镜观察结果,将亚洲玉米螟(Ostrinia furnacalis)幼虫血细胞分为粒细胞、浆细胞、类绛色细胞、原血细胞和球形血细胞五类。调查了幼虫的血细胞总数(THC)和各类血细胞数量(DHC)的变化情况。从三龄末期到五龄第五天期间,幼虫的THC在蜕皮前后会下降,蜕皮后约12h降到最低点,然后又慢慢回升。在五龄幼虫前5d期间,浆细胞在前第三天呈增加趋势,之后开始下降,而粒细胞呈相反趋势。浆细胞和粒细胞具有附着延展性,它们可以附着在载玻片表面,但延展能力不同。血细胞可以迅速黏附在外源物如葡聚糖凝胶珠表面形成包囊,部分包囊会发生黑化现象。体外培养条件下,血细胞也可以形成包囊,其结构与体内形成的包囊差异不大。     

Isolation and characterization of a hemocyte aggregation inhibitor from hemolymph of Manduca sexta larvae

A protein that inhibits hemocyte aggregation has been isolated from hemolymph of Manduca sexta larvae and named hemocyte aggregation inhibitor protein (HAIP). HAIP has a M_r = 50,000, pI = 8.5, and contains 7% carbohydrate. It is present at 230 ± 20 μg/ml in hemolymph of day 3 fifth instar larvae. Antibodies to HAIP do not cross-react with M. sexta hemolin, which is similar in size and charge and also inhibits hemocyte aggregation. HAIP and hemolin have some similarity in amino acid composition and NH₂-terminal sequence, but are different in overall secondary structure, as determined by CD spectroscopy. The concentration of HAIP in hemolymph is not affected by injection of larvae with bacteria. A protein of approximately 50,000 daltons that reacts with antibody to M. sexta HAIP is present in hemolymph of Bombyx mori, Heliothis zea, and Galleria mellonella. Although the function of HAIP in vivo is not yet clear, it may have a role in modulating adhesion of hemocytes during defensive responses. © 1994 Wiley-Liss, Inc.     

Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ

Background

Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster.

Methodology/Principal Findings

To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids.

Conclusions/Significance

From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.     

幼虫密度对草地螟血细胞数量和组成的影响

为了阐明幼虫密度对草地螟Loxostege sticticalis L.（鳞翅目： 螟蛾科）细胞免疫能力的影响, 本研究调查了在活体灰菜植株上1,5,10和20头/瓶（900 mL）4种密度条件下的其5龄幼虫血细胞种类、数量和组成。结果表明： 草地螟幼虫血淋巴中有原血细胞、浆血细胞、 颗粒血细胞、珠血细胞和类绛色血细胞等5种（类）血细胞。血细胞总数、 浆血细胞、颗粒血细胞数量随幼虫密度的增加而显著递增, 但原血细胞、珠血细胞和类绛色血细胞数量在幼虫密度间的差异不明显;各种血细胞所占血细胞总数的比例在4个密度中的排序相同, 但10和20头/瓶密度下的浆血细胞比例显著高于1头/瓶的,其余4种血细胞的比例在不同密度之间无显著差异。可见, 幼虫密度主要是通过影响草地螟幼虫浆血细胞和颗粒血细胞的数量及血细胞总数, 从而影响草地螟的细胞免疫能力。     

Hemocytes of Leiobunum limbatum and two other species of harvestmen (Arachnida,Opiliones): Morphological classification and functional aspects

The hemocytes of Leiobunum limbatum, Mitopus morio, and Opilio ravennae number from about 8,000 (juveniles) to 41,000 (pregnant females) per microliter of hemolymph. Five different types of hemocytes occur in all three species and both sexes. According to their ultrastructural appearance and their similarities to other arthropod hemocytes these five types are designated as prohemocyte, plasmatocyte, granulocyte, coagulocyte, and spherulocyte. From the ultrastructural point of view the prohemocytes are interpreted as stem cells for plasmatocytes which on their part differentiate into granulocytes. Transitional stages which would indicate the origin of coagulocytes and spherulocytes could not be found. Granulocytes and spherulocytes are interpreted as being storage cells; coagulocytes burst when hemolymph is transferred to a microscopic slide. Plasmatocytes are involved in the removal of dead cells or cell fragments. Plasmatocytes are demonstrated as being able to phagocytize and digest bacteria.     

Characterization of the Hemocytes in Larvae of Protaetia brevitarsis seulensis: Involvement of Granulocyte-Mediated Phagocytosis

Hemocytes are key players in the immune response against pathogens in insects. However, the hemocyte types and their functions in the white-spotted flower chafers, Protaetia brevitarsis seulensis (Kolbe), are not known. In this study, we used various microscopes, molecular probes, and flow cytometric analyses to characterize the hemocytes in P. brevitarsis seulensis. The circulating hemocytes were classified based on their size, morphology, and dye-staining properties into six types, including granulocytes, plasmatocytes, oenocytoids, spherulocytes, prohemocytes, and adipohemocytes. The percentages of circulating hemocyte types were as follows: 13% granulocytes, 20% plasmatocytes, 1% oenocytoids, 5% spherulocytes, 17% prohemocytes, and 44% adipohemocytes. Next, we identified the professional phagocytes, granulocytes, which mediate encapsulation and phagocytosis of pathogens. The granulocytes were immunologically or morphologically activated and phagocytosed potentially hazardous substances in vivo. In addition, we showed that the phagocytosis by granulocytes is associated with autophagy, and that the activation of autophagy could be an efficient way to eliminate pathogens in this system. We also observed a high accumulation of autophagic vacuoles in activated granulocytes, which altered their shape and led to autophagic cell death. Finally, the granulocytes underwent mitotic division thus maintaining their number in vivo.     

Hemocyte differentiation in the hematopoietic organs of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>: prohemocytes have the function of phagocytosis

Hemocytes isolated from the larval hematopoietic organs of the silkworm were classified following staining with acridine orange and propidium iodide. Among the hemocytes isolated from the hematopoietic organs of whole fifth larval and wandering stages, most were prohemocytes (60%–70%) and oenocytoids (30%–40%). Granulocytes comprised only about 0.5%–1% at the wandering stage and were even rarer at other stages; no spherulocytes or plasmatocytes were found. Therefore, hemocyte differentiation inside larval hematopoietic organs is not as extensive as previously thought. Following 10–30 min in vitro culture of hemocytes isolated from larval hematopoietic organs, many young granulocytes and plasmatocytes appeared. Furthermore, during phagocytosis assays, prohemocytes were seen to adopt the morphology of plasmatocytes, containing fragments of phagocytosed cells. Our results underline the similarities between Drosophila and Bombyx hematopoiesis.     

Losing the battle against fungal infection: suppression of termite immune defenses during mycosis

The dampwood termite, Zootermopsis angusticollis is known to generate humoral immune responses to the entomopathogenic fungus Metarhizium anisopliae. However, little is known about how the termite's cellular immune system reacts to fungal infection. To test the effect of conidia exposure on cellular immunity, we quantified the number and types of hemocytes in the hemolymph of naïve nymphs and compared their circulating counts with those of nestmates exposed to 0, 2 × 10³, 2 × 10⁶ or 2 × 10⁸ conidia/ml doses. These termites were then bled and their hemocytes counted on days 1, 2, 3, 4, 7 post-exposure. Our results show, first, that naïve Z. angusticollis nymphs have three different blood cell types tentatively identified as granular hemocytes, prohemocytes and plasmatocytes. In these individuals, plasmatocytes were on average 13.5 and 3.3 times more numerous than granular hemocytes and prohemocytes, respectively. Second, a full factorial general linear analysis indicated that hemocyte type, time elapsed since conidia exposure and conidia dosage as well as all their interactions explained 43% of the variability in hemocyte density. The numbers of prohemocytes and particularly plasmatocytes, but not granular hemocytes, appear to be affected by the progression of disease. The decline in hemocyte numbers coincided with the appearance of hyphal bodies and the onset of “sluggish” termite behavior that culminated in the insect's death. Hemocyte counts of infected males and females were affected to the same extent. Hence, M. anisopliae overtakes the cellular immune responses of Z. angusticollis mainly by destroying the host's most abundant hemocyte types.     

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae’s investment into the different types of hemocytes.     

Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera,Carabidae)

The present study focuses on the ability of Pterostichus melas italicus Dejean to mount cellular and humoral immune responses against invading pathogens. Ultrastructural analyses revealed the presence of five morphologically distinct types of hemocytes: prohemocytes, plasmatocytes, granulocytes, oenocytoids and macrophage-like cells. Differential hemocyte counts showed that plasmatocytes and granulocytes were the most abundant circulating cell types and plasmatocytes exhibited phagocytic activity following the latex bead immune challenge. Macrophage-like cells were recruited after the immune challenge to remove exhausted phagocytizing cells, apoptotic cells and melanotic capsules formed to immobilize the latex beads. Total hemocyte counts showed a significant reduction of hemocytes after latex bead treatment. Phenoloxidase (PO) assays revealed an increase of total PO in hemolymph after immune system activation with lipopolysaccharide (LPS). Moreover, the LPS-stimulated hemocytes showed increased protein expression of inducible nitric oxide synthase, indicating that the cytotoxic action of nitric oxide was engaged in this antimicrobial collaborative response. These results provide a knowledge base for further studies on the sensitivity of the P. melas italicus immune system to the environmental perturbation in order to evaluate the effect of chemicals on non-target species in agroecosystems.     

The hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae)

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.     

Changes in hemocytes of Plutella xylostella after parasitism by Diadegma semiclausum

We examined the changes of hemocytes in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), due to parasitism by the endoparasitoid Diadegma semiclausum (Hymenoptera: Ichneumonidae). Necrosis of prohemocytes in different stages was observed while cell death was absent in the mature hemocytes in the parasitized larvae, which was related to the declined total hemocyte count per microliter (THC). THC in the host hemolymph declined sharply by 12 h post-parasitization and then remained at a low level. When hemocytes of the parasitized larvae were cultured in vitro, encapsulation ability was suppressed coincidently with the inhibited spreading ability; however, such effects were transient. Simultaneously, activation of the prophenoloxidae from the hemocytes was inhibited. Unlike the results of previous studies, the decrease in hemocytes, which was due to the necrosis of the prohemocytes instead of the mature hemocytes in our study, was not responsible for the impaired encapsulation. Our studies suggest that parasitism by D. semiclausum have some effects on hematopoietic regulation and on hemocyte immune reaction of P. xylostella larvae.     

Hemocytes of the blowfly Calliphora vicina and their dynamics during larval development and metamorphosis

On the basis of in vitro observation of live cells and examination of stained slides of larval and prepupal Calliphora vicina hemolymph, seven types of hemocytes have been detected: prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes, and plasmatocytes I-IV, a. The last representing sequential stages of one cell line differentiation. Prohemocytes are basic cells, from which other forms of hemocytes derive outside the hemopoietic tissue, i.e. in free hemolymph. At the last larval instar, three waves of hemopoiesis occur. Either wave tends to increase the general number of cells and to change the quality of hemocyte population. The first wave occurs at the close of larva feeding and is accompanied by increase in the number of hyaline hemocytes, thrombocytoids and larval plasmatocytes. The second wave of hemopoiesis occurs after the larva's crop emptying. In this period the main increase of hemocyte population occurs at the expense of prohemocytes and plasmatocytes I. The most significant (five-fold) explosion of the population of free hemocytes takes place at the onset of pupariation and correlates with the rise of ecdysone titer. At the first stage of this peak, the amount of plasmatocytes I sharply increases. Further on these are rapidly differentiated into plasmatocytes II and III. After the puparium formation, hemocytes are reduced in number. Plasmatocytes III phagocytose fragments of destroyed larval tissues, pass to the stage of plasmatocytes IV (macrophages), and partially settle on tissues.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.     

Integument and hemocyte peptides

There are four routing classes of integument peptide in the caterpillar of Calpodes ethlius. The epidermis secretes peptides apically into the cuticle (C), basally into the hemolymph (H) and in both directions (BD). Peptides in a 4th class (T), are presumed to be transported across the epidermis, because the epidermis does not synthesize them although they occur in both cuticle and hemolymph. In a search for the origin of the presumed transepidermal peptides we found that hemocytes contain some peptides from all four routing classes. Peptides prepared from washed hemocytes reacted in immunoblots to antibodies against integument peptides prepared from hemolymph and cuticle. These peptides are probably synthesized by hemocytes because they matched those from medium containing [³⁵S]methionine in which hemocytes had been incubated. Calpodes hemolymph contains four hemocyte types. Immunogold labelling localized integument peptides in the secretory pathway of granulocytes and spherulocytes and in the cytosol of oenocytoids but not in plasmatocytes. Each peptide was localized in a particular kind or kinds of hemocyte. Granulocyte secretory vesicles reacted with antibodies to C180, C55 and BD82 kDa peptides. Spherulocytes secretory vesicles reacted with antibodies to C180, C55, BD89, BD82 and a 78 kDa peptide presumed to be the precursor of T66. Oenocyotoids reacted with antibodies to H45, 38, 32, 23 and BD89 kDa peptides. Spherulocytes were the only tissue to react with antibodies to the T66 kDa peptide that is found abundantly in cuticle and hemolymph. Spherulocytes are therefore presumed to secrete the 66 kDa peptide into the hemolymph from where it is transported to the cuticle. The C180 and C55 kDa peptides do not occur in hemolymph. Their presence in granulocytes and spherulocytes may be associated with hemocyte functions such as basal lamina formation, since immunogold localized them in that part of the basal lamina next to the hemolymph, as would be expected if hemocytes deposited components onto the exposed hemolymph surface. The presence of hemolymph peptides in oenocytoids is more difficult to interpret, since the antigenic reactions are localized in the cytosol rather than in the secretory pathway expected for exported proteins. We conclude that integument peptides are not secreted only by the epidermis, nor is the cuticle their only destination.     

Hematopoiesis in Hematopoietic Organ in Euprepocnemis shirakii (Orthoptera, Insecta)

ABSTRACT The present study was undertaken to investigate the hematopoiesis of the hematopoietic organ found in orthopteran Euprepocnemis shirakii under both light and transmission electron microscopy. It was shown that the hemocyte differentiation of E. shirakii was distinct from features compared to patterns previously reported in other insect species: Their prohemocytes, plasmatocytes, granulocytes I, granulocytes II and spherulocytes were all derived from the hematopoietic stem cells surrounded by reticular cells. This pattern of hematopoiesis was also first observed in the Orthoptera. Thus, findings of this study strongly suggested that the patterns of hematopoiesis in insects differed, among groups, showing the need of an extensive investigation in order to correctly comprehend the patterns of hemocyte differentiation in insects in accordance with first order, and then systematically important taxa.     

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

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 species^1-5, as well as the wealth of information available regarding the insect''s immune system^6-8, 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 microscopy^{13, 14}.