Insect immune responses to multiple pathogen groups including viruses, bacteria, fungi, and entomopathogenic nematodes have traditionally been documented in model insects such as Drosophila melanogaster, or medically important insects such as Aedes aegypti. Despite their potential importance in understanding the efficacy of pathogens as biological control agents, these responses are infrequently studied in agriculturally important pests. Additionally, studies often neglect to investigate responses against different pathogen groups, and typically focus on only a single time point during infection. As such, a robust understanding of immune system responses over the time of infection is often lacking. This study was conducted to understand how 3^rd instar larvae of the major insect pest Helicoverpa zea responded over time to infection by four different pathogenic groups: viruses, bacteria, fungi, and entomopathogenic nematodes. Physiological immune responses were assessed at 4-, 24-, and 48-hours post-infection by measuring hemolymph phenoloxidase concentrations, hemolymph prophenoloxidase concentrations, hemocyte counts, and encapsulation ability. Transcriptional immune responses were measured at 24-, 48-, and 72-hours post-infection by quantifying the expression of PPO2, Argonaute-2, JNK, Dorsal, and Relish. This gene set covers the major known immune pathways: phenoloxidase cascade, siRNA, JNK pathway, Toll pathway, and IMD pathway. Our results indicate H. zea has an extreme immune response to Bacillus thuringiensis bacteria, a mild response to Helicoverpa armigera nucleopolyhedrovirus, and no detectable response to either the fungus Beauveria bassiana or Steinernema carpocapsae nematodes.

I. Pathogens

Helicoverpa zea caterpillars were purchased from Benzon Research Inc. (Carlisle, PA) as eggs and were reared on artificial diet purchased from Southland Products Inc. (Lake Village, AR) until reaching the targeted instar. Larvae were maintained in rearing chambers under a constant temperature of 25°C, relative humidity of 70%, and light-dark ratio of 14:10 for all experiments. The strain of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) was provided by AgBiTech LLC (Fort Worth, TX), and is listed under the trade name Heligen®. To get to the desired concentration for inoculation, the highly concentrated viral solution was serially diluted from 7.5 × 10⁹ occlusion bodies/mL to 7.5×10⁵ occlusion bodies/mL based on previous research (Black et al. unpublished). The Beauveria bassiana strain GHA was isolated from BotaniGard Maxx® purchased from BioWorks Inc. (Victor, NY). The spores were extracted by placing 30mL of the solution into a 50mL Falcon tube and centrifuging at 3000 rpm for 10 minutes, then removing the supernatant and adding 30mL of sterile water. The solution was then vortexed, and this process was repeated three times before the final pellet was resuspended in sterile water and stored at 4°C until needed. Spore suspension viability was tested prior to use by making a serial dilution and plating the 5^th and 6^th dilution. The plates were allowed to incubate at room temperature for three days and then colonies were counted and multiplied by the dilution factor to determine viable spore concentration in the spore suspension. The Bacillus thuringiensis pathogen was diluted from Thuricide BT® purchased from Southern AG Insecticides, Inc. (Hendersonville, NC) to 7.5×10⁵ CFUs/mL. Steinernema carpocapsae was purchased from ARBICO Organics (Oro Valley, AZ), and serially diluted to 7.5×10⁵ nematodes/mL.

i. Procedures

Once H. zea larvae molted to 3^rd instar, they were inoculated with one of five treatments determined prior to initiation of the experiment. This was done by pipetting a 10µL drop of liquid containing either the treatment pathogen dosage or a control of sterile deionized water onto a fresh piece of artificial diet, approximately 50mg, where it was absorbed. Inoculation time zero was defined as this point of pathogen introduction into the individual larva’s environment, which was a sterilized 2oz deli cup (ULINE, Pleasant Prairie, WI). The larvae were then allowed to feed on the infested diet and only those that consumed the inoculated diet cube were utilized in each of the two experiments described below. Although oral infection is not the primary infection route for entomopathogenic fungi or nematodes, they are still capable of infecting through the midgut [72, 73]. Also, the surface contamination of the diet should not inhibit the potential for infection through the insect cuticle or other orifices, but would simulate a more likely environment when used as a foliar bio-pesticide [74].

xi. 1: Temporal Physiological Immune Response

This experiment utilized five pathogen treatments (Control, HearNPV, B. bassiana, B. thuringiensis, and S. carpocapsae), and subdivided each treatment into sample times. Three temporal sampling points of 4 hours, 24 hours, and 48 hours post-inoculation were implemented to develop an understanding of how the H. zea immune response changes during a pathogen invasion. Later temporal sample points were not possible because most larvae succumbed to the pathogens by three days and survivors across all pathogens were too few for meaningful analysis. Individual larvae were sampled by extracting their hemolymph at the designated sample times as described below. Hemolymph from two larvae were pooled to make one biological replicate. Each pathogen × time-treatment had 32-34 biological replicates collected across three independent trials.

h. Extraction

Hemolymph extraction occurred by sterilizing the larva with an ethanol wash, weighing the larva, and then chilling the larva on ice before piercing the larva with a sterile 27-gauge needle between the second pair of prolegs. The insect hemolymph was allowed to drain directly into an Eppendorf tube on ice and placed into a -20°C freezer immediately upon completion of the extraction. While extracted volumes varied across biological samples, each sample had at least 70μL to complete all the physiological assays described below.

h. Phenoloxidase and Prophenoloxidase Assay

An 8µL aliquot of hemolymph was added to 360µL of sodium cacodylate (NaCac) in a 2mL microcentrifuge tube. The sample was then evenly divided into two 2mL microcentrifuge tubes. One tube had the prophenoloxidase (PPO) activated by adding 20μL of 20mg/mL chymotrypsin suspended in NaCac buffer, while the other tube served as the spontaneously activated phenoloxidase (PO) control with 20μL NaCac added. Samples were incubated at 25°C for one hour to allow the PPO time to be activated prior to microplate reader analysis. All analyses were run in duplicate using Costar® 96 well flat bottom plates and analyzed in an Infinite M200 Pro microplate reader (Tecan, Mӓnnedorf, Switzerland). Plates were first loaded with 90μL of the sample solution per well, and then 90μL of 4mM dopamine was pipetted into each well. Once all wells had both the sample solution and dopamine, the plate was placed into the microplate reader and the absorbance was measured at 492nm. The amount of phenoloxidase in the sample was calculated in phenoloxidase units, where one unit is the amount of enzyme required to increase the absorbance by 0.001 per minute.

h. Protein Assay

Protein was measured using a BCA Protein Assay Kit II (BioVision Inc., Milpitas, CA) by adding 25µL of the hemolymph solution to 200μL of the BCA working reagent in each of the Costar® 96 well flat bottom plate wells. The plate was covered and incubated at 37°C for 30 minutes. After the incubation the absorbance was measured at 562nm. A standard curve using the provided standards was utilized to determine protein concentration (μg/mL). Once protein concentrations were known, phenoloxidase units were expressed as phenoloxidase units per mg of protein.

h. Count

Hemocyte counts were determined using an improved Neubauer hemocytometer. The hemocytometer was loaded with 8µL of pure hemolymph, allowed to settle for 20 minutes and the five non-adjacent squares were counted on each side of the hemocytometer to give an estimate of hemocyte density.

liii. Activity Assay

Lytic activity against the bacterium Micrococcus lysodeikticus was determined using a lytic zone assay. Agar plates were made prior to the assay by mixing 10mL of agar suspension containing the following: 1.5g agar, 0.75g M. lysodeikticus in 50mL 0.2 M potassium phosphate buffer, 0.1mg/mL streptomycin sulphate, and 67mM potassium phosphate buffer (pH 6.4) and pouring the mixture into a plastic petri dish and stored in a 4°C refrigerator. For each plate, approximately 13 holes with a diameter of 2mm were punched into the agar and filled with 1µL of hemolymph, with two technical replicates per sample. The plates were incubated at 32˚C for 24 hours, photographed, and the diameter of the clear zones calculated with ImageJ imaging software. Standard curves were obtained using a serial dilution of egg white lysozyme, and concentration of egg white lysozyme equivalents were calculated. Standard curves were developed for each batch of plates. Based on the logarithmic connection to lysozyme concentration, diameters of lytic zones obtained from the hemolymph samples were converted to HLAs (ng/µL – equivalents of hen egg white lysozyme activity).

li. Response Assay

Immediately after the hemolymph extraction, a 3mm long piece of nylon monofilament was inserted completely into the puncture wound of each larva in such a way to minimize the potential of rupturing the midgut. Surviving larvae were returned to diet for 24 hours. After that time, the surviving larvae were frozen and upon death the nylon monofilament was dissected out, mounted on a slide and photographed. The level of melanization and area of cell cover was quantified using ImageJ [68] imaging software distributed by Fiji [69]. One larva in each pooled biological sample was subjected to an encapsulation assay. If the gut was ruptured or the nylon filament was not recovered during the dissection, these larval samples were discarded.

lii. Analysis

Pair-wise MANOVAs were conducted for each Pathogen × Control pairing, with Treatment and Time as main effects, and the measured immune responses as dependent variables. The average pooled larval weight was used as a covariate since all dependent variables were analyzed for all samples. Pillai’s trace statistic was used to compare differences from the Control. Then, each immune response was subjected to an ANOVA and Tukey’s HSD. All data were checked for conformity and normalcy. All analyses were conducted in R Studio [70].

xi. 2: Temporal Transcriptional Immune Response

This experiment utilized the same five pathogen treatments as in Experiment 1, but each treatment was subdivided into three different sample times: 24 hours, 48 hours, and 72 hours post-inoculation. Changes in the expression of genes involved in the major immune pathways were measured as opposed to physiological immune responses. Twenty-five larvae were reared for each pathogen × sample time treatment combination. Five larvae were pooled for each biological replicate, resulting in five biological replicates per pathogen x sample time treatment combination. Hemolymph was extracted as described above, except immediately following extraction, the 2mL microcentrifuge tubes were flash frozen in liquid nitrogen then stored in a -80°C freezer. RNA was extracted from the hemolymph samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentrations were determined by using a NanoView Plus (General Electric, Boston, MA). RNA concentrations were then standardized to 100ng/μL before being converted to cDNA using iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). The resulting DNA concentrations were determined with a NanoView Plus, and diluted to 100ng/μL by adding RNase and DNase free water. Once sample DNA concentrations were standardized, Quantitative Real-Time PCR (qPCR) was conducted using primers targeting specific immune genes, Actin as a housekeeping gene and RPS3 as a verification gene that Actin was not differentially expressed across treatments (Table 1). Transcript-specific primers were designed by first extracting putative transcript sequences from the published H. zea draft genome and associated annotation file using gffread [71, 75]. This generated a sequence file of parsed mRNA and coding sequences (CDSes) from which we ran BLASTn searches using Helicoverpa armigera CDSes of Actin, RPS3, PPO2, Argonaute-2, JNK, Dorsal, and Relish as the query. The obtained H. zea transcripts were then secondarily validated through BLAST searches of the NCBI database to confirm sequence identifications. The obtained transcripts were passed through the PrimerQuest™ Tool provided by Integrated DNA Technologies to generate qPCR primers. Conventional PCR products were obtained from each primer pair and purified using a Monarch DNA Gel Extraction Kit (T1020S) and submitted for Sanger Sequencing to validate their specificity to the desired transcripts. qPCR was conducted using SYBR™ Green (Bio-Rad Laboratories, Hercules, CA) and Precision Blue Real-Time PCR Dye (Bio-Rad Laboratories, Hercules, CA) in a C1000 Touch Thermal Cycler with the CFX384 Real-Time System attachment (Bio-Rad Laboratories, Hercules, CA). Data was then exported into CFX Maestro (Bio-Rad Laboratories, Hercules, CA) software, and analyzed using ANOVA and Tukey’s HSD post hoc test in R Studio (R Core Team, 2020). All target genes were previously determined to be differentially expressed during pathogenic infection in H. armigera and S. frugiperda, two species closely related to H. zea [48, 76].