Humoral immune responses differ according to antigen in Children’s pythons (Antaresia childreni)

Humoral immunity provides a robust defense against pathogens through the production of antibodies with diverse functions, yet it remains poorly understood in ectotherms. In this study, we compared the extent and time course of the humoral immune response to two commonly used non-pathogenic antigens in male and female Children’s pythons (Antaresia childreni). Two separate experimental cohorts of adult snakes from the same colony were used, each consisting of 10 males and 10 females. One cohort received either lipopolysaccharide (LPS) or a control injection, while the other cohort received keyhole limpet hemocyanin (KLH) or a control injection. Blood samples and blood smears were collected prior to treatment and at set intervals following injection. Humoral immune responses were assessed by indirect ELISAs for antigen-specific IgY. White blood cell differentials were performed on stained blood smears to evaluate the early innate immune response. KLH injections elicited a robust anti-KLH IgY response, increasing from day 14 through day 100 and remaining elevated through day 183. Additionally, males had higher anti-KLH IgY titers than females at days 14 and 21. In contrast, LPS injections failed to cause IgY production throughout the time frame of the study. Leukocyte responses were only observed following KLH injections, with increased monocyte counts at day 7. These results demonstrate that KLH may be a more effective antigen for studies investigating humoral responses in reptiles due to its novelty to these animals. In contrast, snakes likely had variable prior exposure to LPS-like antigens, since they are in the walls of gram-negative bacteria, which are ubiquitous. Overall, our results provide important context for understanding the variability of the humoral immune time course in reptiles.

Animals face a variety of pathogens, with many epizootic infections becoming more prevalent and widespread due to ongoing anthropogenic environmental changes (Fèvre et al., 2006). To combat these invaders, all animals possess some form of immune system to recognize antigens and destroy pathogens. In jawed vertebrates, the rapid nonspecific innate immune system and slower but antigen-specific adaptive immune system are activated by different antigens through separate pathways (Flajnik and Kasahara, 2010). Innate immune cells, specifically antigen-presenting macrophages and dendritic cells, are key to both pathways as they are responsible for the initial innate immune response once an antigen has entered the body while also contributing to the activation of the subsequent adaptive response (Rios and Zimmerman, 2015). During the early stages of the immune response, macrophage and monocyte activity increases to phagocytize pathogens, heterophils are recruited to promote inflammatory responses, and dendritic cells process and present antigens to lymphocytes to activate them (Janeway et al., 2001).

The humoral immune system, one branch of the adaptive immune system, is characterized by antibody production by B cells and responds to exogenous antigens. Activation of B cells of the humoral immune system occurs via two pathways, determined by whether T cell help is required (Janeway et al., 2001). Thymus-independent (TI) antigens, usually consisting of polysaccharides and lipids, bind directly to B cells and stimulate antibody production (Stein, 1992). TI antigens, such as lipopolysaccharide (LPS) from the cell membranes of gram-negative bacteria, can trigger rapid but lower magnitude increases in antibody titers (Janeway et al., 2001). Thymus-dependent (TD) antigens, which include proteins, viral RNA, and parasites, require T helper cells to trigger B cell maturation and antibody production (Stein, 1992). TD antigens, such as the respiratory protein keyhole limpet hemocyanin (KLH) from the giant keyhole limpet (Megathura crenulata), produce slower responses but typically have higher peak antibody titers in mice (Kao et al., 2017). However, stronger responses to TD antigens are not ubiquitous across taxa, as work in fish has shown stronger humoral responses to TI antigens (Granja et al., 2019). Thus, while differences in B cell activation and antibody production triggered by TI and TD antigens are well understood in mammalian models, these pathways may differ in other, less studied taxa such as reptiles.

The humoral immune system of reptiles differs from mammalian models in several ways. The initial humoral response in both groups is characterized by IgM production, followed by isotype switching to IgG in mammals and the homologous IgY in reptiles (Pettinello and Dooley, 2014; Matz and Dooley, 2023). The initiation of antibody production and the time to reach peak titers is much slower in reptiles, reaching peak titers after several weeks compared to approximately two weeks in mammals (Zimmerman et al., 2010). This difference may be due in part to reptiles being ectotherms, and therefore having lower metabolic rates compared to endothermic animals. The process of affinity maturation, whereby B cells with high affinity for the antigen are selected and subsequently proliferate, and the formation of immunological memory through the production of long-lived memory cells are thought to be reduced or completely absent in reptiles (Zimmerman et al., 2010; Sandmeier et al., 2012). This likely results from the lack of germinal centers, which facilitate interactions between B cells and T cells and the maturation of B cells, in reptiles and other ectothermic taxa (Hsu, 1998). Additionally, as in mammals and other systems, there are sex-based differences in the humoral immune responses of reptiles, especially during times of reproductive investment (Roved et al., 2017). Females may produce faster and more robust humoral responses, potentially due to estrogen promoting B cell function while testosterone can suppress immune function, although this difference may be less pronounced during non-reproductive periods (Fink and Klein, 2018; French et al., 2022). Stronger humoral responses in females have been observed in a species of snake (Psammophis sibilans) and turtle (Chrysemys picta) (Saad and Shoukrey, 1988; Judson et al., 2020), but other work has found no sex-differences in humoral immunity in species of snake (Liasis fuscus), lizard (Salvator merianae), and turtle (Trachemys scripta, Gopherus agassizii) (Ujvari and Madsen, 2011; Sandmeier et al., 2012; Zimmerman et al., 2013b; Mestre et al., 2017). As sex-based differences in immunity, and humoral immunity overall, varies among taxa, further study is needed, particularly across squamate reptiles, which account for more than 96% of reptile species (Pincheira-Donoso et al., 2013).

In this study, we assessed differences in the humoral immune response to two commonly used antigens in immunological research, LPS, a TI antigen, and KLH, a TD antigen, through measuring antigen-specific IgY using non-reproductive adult Children’s pythons, Antaresia childreni. The small adult body size of this species, compared to other pythons, makes them practical for laboratory housing in large numbers while still being large enough to permit repeated blood sampling. Existing literature on this species has explored their innate immune responses, although there is limited information on humoral immunity (Brusch et al., 2017). To capture differences in the initial innate immune response, we examined white blood cell (WBC) differentials. We hypothesized that WBC differentials should differ between the two antigens, with LPS promoting a greater heterophil response due to its activation of pro-inflammatory pathways, and that differences between sexes in this response should be minimal. For antigen-specific IgY, we examined differences between males and females in the time course and magnitude of the response. We hypothesized that the kinetics of the humoral response will differ between LPS and KLH, with LPS producing a faster but lower magnitude IgY response and KLH producing a slower but more robust response. Additionally, since LPS is a ubiquitous antigen in the environment, we expected that IgY responses will vary greatly among individuals due to differences in past exposure, whereas the completely novel KLH should lead to more consistent IgY production. As the animals in our study were nonreproductive, we expected there would be minimal sex-based differences in the humoral immune response.

Animal care and maintenance

Children’s pythons, Antaresia childreni, used in this study are part of an existing colony at Arizona State University (ASU), Tempe, AZ, USA. For logistical reasons, snakes receiving a KLH immune challenge were housed at ASU while snakes receiving an LPS immune challenge were housed at Utah State University, UT, USA. All snakes used were between 10–17 years of age at the time of the study. Sampling for LPS snakes was conducted in 2024 and sampling for KLH snakes was conducted in 2024-2025. Following the conclusion of sampling, snakes were retained as part of the larger colony for use in future studies.

Snakes were housed individually in cages (30 × 45 × 15 cm, Freedom Breeder, Turlock, CA) that were kept at a room temperature of 29 ± 1 °C with access to a thermal gradient of 29-45 °C provided by an under-cage heating element. The rooms had a 12L:12D light cycle (lights on at 0800 hr). Snakes were fed previously frozen laboratory mice at a frequency to maintain a healthy body condition and provided water ad libitum. At each location, 20 snakes were assigned to either the control (n = 10) or immune challenge condition (n = 10) using stratified random sampling based on mass and sex. For each location, control and immune challenge groups had equal sex ratios and there were no significant differences in initial animal mass (KLH: KLH-treated mass = 579 g ± 53 g, control mass = 560 g ± 70 g; LPS: LPS-treated mass =537 g ± 65 g, control mass = 505 g ± 67 g). All animal procedures were approved under Arizona State University Institutional Animal Care and Use Committee protocol #23-1948R or Utah State University Institutional Animal Care and Use Committee protocol #13172.

Immune challenge

For all snakes, baseline blood samples were collected via cardiocentesis, and this was immediately followed by an intracoelomic injection of an immunogen or phosphate buffered saline (PBS, as a vehicle control). KLH-treated snakes received a dose of 4 µg KLH (374825, Sigma-Aldrich)/1 g snake mass at a concentration of 2.5 mg KLH/1 mL (e.g., a 500 g animal received a 0.80 mL injection). LPS immune challenges were given at a dose of 15 µg LPS (L3129, Sigma-Aldrich)/1 g snake mass at a concentration of 6.0 mg LPS/1 mL (e.g. a 500 g animal received a 1.25 mL injection), the dosage of LPS showing detectable humoral immune responses in green iguanas (Iguana iguana) (Ki et al., 2024). KLH injection preparation was based on work in Siberian hamsters (Phodopus sungorus; French et al., 2016). However, the dosage was slightly increased to a higher concentration to ensure humoral responses as reptiles tend to be less responsive than mammals. The route of injection was based on previous studies that showed strong responses to intracoelomic injections in reptiles (Hudson et al., 2021; Ki et al., 2024). No adjuvant was used with LPS nor KLH, as adjuvants may have variable effects on the immune response and previous work has shown strong responses to injections without adjuvant (Hellebuyck et al., 2014; French et al., 2016; Lind et al., 2020; Hudson et al., 2021; Ki et al., 2024). Control snakes received an equivalent mass-adjusted volume injection of sterile PBS. Additional blood samples (≤ 500 µL) were taken from the LPS-treated snakes and their counterpart control snakes at 7, 21, 35, and 81 days post injection. For logistical reasons, KLH-treated snakes and their controls were sampled on a modified timeline of 7, 14, 21, 100, and 183 days post injection. Immediately after collection, blood samples were centrifuged at 6000 rpm for 5 minutes to separate plasma from blood cells, and plasma was placed into a separate microcentrifuge tube and frozen at -80 °C. At each sampling time point, thin blood smears using roughly 5 µL of whole blood were prepared and allowed to air dry at room temperature before being fixed in methanol.

Immunological assays

KLH-specific IgY antibodies were measured through an enzyme-linked immunosorbent assay (ELISA) adapted from Ki et al. (2024). Briefly, a 96-well plate was coated with 200 µL of a solution of 0.5 mg KLH/mL of 0.1 M bicarbonate buffer (pH 7.2) and incubated overnight at 4 °C. The plate was washed and blocked using milk powder dissolved in a PBS and TWEEN 20 solution. Following plate preparation, plates were stored at -20 °C. The assay was performed using 100 µL of 1:16 plasma diluted in 1% milk powder pipetted in duplicate and then incubated at 37 °C for three hours. Plates were then washed and 150 µL rabbit-anti-iguana-IgY antibodies conjugated with HRP (SouthernBiotech, custom antibody, SBCS-58, Birmingham, AL) diluted 1:1000 in 1% milk powder solution was added. Plates were incubated at 37 °C for 1 hour, after which 100 µL of TMB substrate was added, and the plates were incubated in the dark for 15 minutes. Following incubation, 50 µL of stop solution was added, and the absorbance was read at 450 nm (xMark; Bio-Rad, California, USA). To calculate IgY concentrations, the absorbance of non-specific binding wells was subtracted from the absorbance of each sample well, and the subsequent absorbance value was converted to concentration using the Beer-Lambert Law (path length = 8 mm, extinction coefficient = 1.4 mg/mL) as done in Ki et al. (2024). Coefficients of variation (CV) were calculated by taking the CV of each of the sample replicates and averaging across all samples within a plate (intra-assay) and between plates (inter-assay). The intra-assay CV was 10.9% for the first assay and 13.9% for the second assay, and inter-assay CV was 12.4%.

LPS-specific IgY antibodies assays followed the above protocol with few alterations. Plates were coated with 5 µg LPS/ml of 0.15 M carbonate buffer (pH 9.6) and incubated and blocked the same as the KLH plates. The assay was performed using 100 µL of 1:10 plasma diluted in 1% milk powder, pipetted into plates in duplicate and incubated overnight at 4 °C. Plates were then washed, and 150 µL rabbit-anti-iguana-IgY antibodies conjugated with HRP (SouthernBiotech, custom antibody SBCS-58, Birmingham, AL) diluted 1:1000 in 1% milk powder solution was added. Plates were incubated at 37 °C for 1 hour, after which 100 µL of TMB substrate was added, and the plates were incubated in the dark for 25 minutes. Following incubation, 50 µL of stop solution was added, and the absorbance was read at 450 nm. The calculation of CV was not possible for this assay because most anti-LPS IgY absorbance values were close to 0, resulting in mathematically inflated values. Validations were conducted for both KLH-specific and LPS-specific IgY ELISAs using serial dilutions of pooled samples from antigen-injected animals at endpoints for each antigen (Supplementary Figures 1A, S1B).

Blood smears were stained in 1:20 modified Giemsa stain (48900-F, Sigma-Aldrich) for 20 minutes and washed in two deionized water baths for 5 minutes. Slides were then left to dry for at least 2 days before counting blood cells. To measure innate immunity, white blood cell differentials were performed on each slide by capturing images of non-overlapping microscope fields including at least 500 cells at 400x (adapted from French et al., 2008) using a Nikon Eclipse 80i microscope and NIS Elements F software (Nikon Instruments Inc., Melville, NY). Cells in these images were counted and identified, and lymphocytes, heterophils, and monocytes were calculated as a ratio to total erythrocytes to account for any differences in animal hydration. Slides from the KLH study animals were examined by two separate observers while slides from the LPS study animals were examined by one observer.

Statistical methods

All statistical analyses were performed in RStudio v.4.4.0 (R Core Team, 2019) using the following packages: ‘mgcv’v 1.9.1 (Wood, 2011), ‘emmeans’ v1.10.1 (Lenth, 2024), ‘tidyverse’v.2.0.0 (Wickham et al., 2019), and ‘ggplot2’ v.3.5.2 (Wickham, 2016). We tested the effects of immune challenges on IgY concentrations and WBC ratios using generalized additive mixed models (GAMMs) fitted using the mgcv package (Pedersen et al., 2019). Due to inter-assay variation in LPS-specific IgY, values at each time point were centered by subtracting the mean value for that time point from each value to enable better comparison. Separate models were fit for each antigen (KLH, LPS) and each immune measure (IgY, monocyte ratio, lymphocyte ratio, and heterophil ratio).All models included fixed effects of sex, treatment, and their interaction, a smooth function of sampling timepoint for each sex-treatment combination (KLH-treated females, control females, KLH-treated males, and control males) to capture nonlinear immune trajectories (Lamb et al., 2024), and a random smooth of time for each individual to account for repeated sampling. Time was modeled based on timepoint index of sampling (1,2,3, etc.) and not on days from baseline, as we expected rapid changes in immune metrics early on and more stable responses at later time points. This approach accounted for higher degrees of freedom for fitting earlier time points due to more frequent sampling and lower degrees of freedom for later time points. Model fitting used restricted maximum likelihood (REML) to prevent overfitting by shrinking smooth terms to linear if data does not support additional complexity (Wood, 2011; Pedersen et al., 2019). To further account for overfitting due to small sample sizes and test the robustness of our results we fit two additional simplified models, one including only a smooth for treatment and the other including no treatment or sex smooths, the latter of which is essentially a linear model (Supplementary Table 3). Across all three models, KLH treatment had a strong effect on KLH-specific IgY, suggesting that the inclusion of sex did not impact our interpretation of treatment affects. The estimate for the sextreatment interaction had a similar magnitude and direction in all three models, with simplified models having a larger confidence interval than more complex models (Supplementary Table 3). The only major difference was that the model with only a smooth term for treatment had significant sextreatment interactions, whereas in the simpler model without smooth terms this interaction was not significant. The latter model also had a worse fit, suggesting the inclusion of smooth terms is necessary to account for the non-linearity of the relationship. For the remaining analyses, simplification of the models did not influence the significance of the fixed effects. Based on these comparisons, the more complex models provide more explanatory value and a better fit with no substantiative differences to the less complex models. As a result of this and discussion with a statistical consultant, we chose to use the more complex model including smooths for all sex*treatment interactions for our analyses. For models with significant fixed effects, we performed Tukey HSD post-hoc pairwise comparisons between sex and treatment using the emmeans package. Statistical significance was determined based on an alpha of 0.05.

IgY

For KLH-specific IgY, while the fixed effect of sex was not significant, both the fixed effect of treatment and the sex-treatment interaction were significant (Table 1, Figure 1). Smooth terms were significant for KLH-treated females and KLH-treated males, marginally significant for control females, and not significant for control males (Table 1). Pairwise comparisons showed that KLH-treated snakes had higher KLH-specific IgY than controls from days 14-183, and KLH-treated males had higher IgY than females at days 14 and 21 (Table 2). Peak titers in KLH-treated snakes were over seven times greater (0.242 mg/mL) than baseline (0.034 mg/mL). For LPS-specific IgY, there were no significant fixed effects or smooth terms (Table 1, Figure 2).

WBC

For KLH, we found a significant fixed effect of treatment on monocyte ratios. Pairwise comparisons showed that, at day 7, KLH-treated males (p = 0.001) and KLH-treated females (p < 0.001) had higher monocyte ratios than control snakes of the same sex, and that KLH-treated females had higher monocyte ratios than KLH-treated males (p = 0.005). We failed to find significant fixed effects of KLH treatment on lymphocyte and heterophil ratios for KLH as well as for all three ratios in response to LPS treatment., (Supplementary Tables 1, S2). For KLH, there were significant smooth terms for control females, KLH-treated females, KLH-treated males, and a marginally significant smooth term for control males for monocyte ratio. Additionally, there were significant smooth terms for control and KLH-treated males for lymphocyte ratio and a significant smooth term for control females for heterophil ratios (Supplementary Table 1). For LPS, there was a significant smooth term for LPS-treated males for monocyte ratio, ratio, a significant smooth term for LPS-treated females for lymphocyte ratio, and a significant smooth term for control males for heterophil ratio (Supplementary Table 2).

Summary

We examined the time course of components of the immune response in Children’s pythons (Antaresia childreni) following exposure to either LPS or KLH. The humoral immune response, measured by antigen-specific IgY, varied greatly between the two antigens. KLH induced a strong IgY response, with a significant increase in titers by 14 days, continued production through day 100, and a limited but significant decline between days 100 and 183. We also detected sex differences in the early IgY response to KLH, with males exhibiting higher titers than females at 14 and 21 days, although both sexes had comparable peak titers at day 100. LPS, however, failed to elicit any production of IgY over the 81-day sampling period. In contrast to the humoral response, no changes to white blood cell differentials were detected in response to either antigen.

Immune response

We found a substantial IgY response to KLH starting at 14 days post-challenge and continuing through 100 days. The time course of the production of IgY in other reptiles has been seen to range from 3–4 weeks (Coe et al., 1976; Ujvari and Madsen, 2011). Peak titers were over seven-fold greater than baseline, which is comparable to the three-fold increases commonly seen in squamates while being lower than the 100-fold increases typically seen in mammals (Sandmeier, 2024). The strong response to KLH, compared to the lack of response to LPS, is likely due to the novelty of the antigen, as KLH is marine in origin and would never have been encountered by the snakes in this study. The lack of previous exposure means that no KLH-specific antibodies would be present to reduce B cell responses by epitope masking. Additionally, because KLH is a TD antigen, requiring T cell help to activate B cells, the magnitude of the IgY response would be expected to be much greater compared to a TI antigen such as LPS (Janeway et al., 2001; Kao et al., 2017). While IgY levels decreased from 100 days to 183 days, they were still much greater than baseline at the end of the study. In reptiles, antigen-specific IgY has been observed to remain in circulation for significantly longer periods than they do in birds or mammals, with antibodies from the primary response being detected up to 34 weeks after immunization (Origgi et al., 2001).

Interestingly, we also observed that male Children’s pythons mounted a faster antibody response to KLH than did females, with higher IgY at 14 and 21 days, although the timing and the extent of peak titers did not differ between sexes. This is in contrast to some past studies, which demonstrated that female snakes produce a faster and more intense humoral response in response to rat red blood cells (Saad and Shoukrey, 1988). These differences are due in part to estrogen promoting B cell responses, whereas testosterone can modulate and reduce these same responses (Roved et al., 2017). While sex differences in immunity are more pronounced during reproductive periods due to increases in steroid hormones and reproductive investment, these trends have still been observed in nonreproductive animals (French and Moore, 2008). Other studies have demonstrated no sex differences in humoral immunity in snakes with boid inclusion body disease, in snakes injected with KLH, or in LPS-binding antibodies in turtles, suggesting that sex differences in immunity may vary across taxa and according to what antigen was used in a given study (Ujvari and Madsen, 2011; Zimmerman et al., 2013b; Windbichler et al., 2019). The higher production of antibodies early in the humoral response by males could be attributed to energetic trade-offs associated with preparing for reproduction, as seen in male garter snakes having greater lymphocyte proliferation than females (Palacios et al., 2013). These results showing greater humoral responses in males should, however, be interpreted cautiously due to the small sample sizes in this study and the relatively small difference in KLH-specific IgY between the sexes.

In contrast to the KLH results, we found no evidence of B cell activation nor IgY production following the LPS immune challenge. Previous work in this species has shown that LPS exposure causes an initial decline in LPS-specific IgY within two days of injection, but with an eventual increase by day 28 (Emanuel et al. in prep). Juvenile green iguanas (Iguana iguana) have shown a strong IgY response to LPS within 1 week and a peak at 2 weeks (Ki et al., 2024). This lack of antibody production may be the result of our experimental protocol, as we did not use an adjuvant with LPS. Adjuvants have been shown to enhance humoral responses to LPS in mammals (Naseer et al., 2025), but previous studies in reptiles have shown robust innate and adaptive responses to LPS without an adjuvant (Hudson et al., 2021; Ki et al., 2024). Alternatively, the absence of a humoral response to LPS in our study could be caused by existing anti-LPS antibodies or natural antibodies preventing antigen detection by B cells through epitope masking, whereby LPS is removed from circulation before B cells can be activated to produce antigen-specific IgY (Zimmerman, 2022). Other studies in both turtles and pythons have demonstrated a lack of a specific humoral response to an antigen due to epitope masking thought to be carried out primarily by natural antibodies in circulation (Ujvari and Madsen, 2011; Sandmeier et al., 2012; Zimmerman et al., 2013a). Given that LPS is a component of the cell wall of gram-negative bacteria, which are ubiquitous in the environment, it is likely that our snakes had prior exposure to LPS and therefore likely had LPS-specific IgY in circulation prior to our LPS challenge (Mazgaeen and Gurung, 2020). Being juveniles, the green iguanas in the previously mentioned study may have had much more limited exposure to LPS, thus enabling a greater response to the experimental challenge with LPS. Thus, novelty of the LPS antigen may affect the extent of the humoral response (Ki et al., 2024). In addition, LPS is a TI antigen and thus may produce a more rapid, but potentially lower magnitude, IgY response compared to a TD antigen (Kao et al., 2017). As such, snakes may have produced low amounts of IgY in response to LPS which we were unable to detect due to sample size and large variation between individuals (Janeway et al., 2001).

While we expected to see a broad leukocyte response early in the sampling period, only monocytes increased in concentration in KLH-treated snakes at day 7. KLH has been shown to stimulate activation and differentiation of monocytes in human cells through production of cytokines and activation transcription factors involved in immune cell production (Presicce et al., 2008; Yasuda and Ushio, 2016). Whether this same mechanism increases monocytes in reptiles or other taxa would require further exploration. The lack of leukocyte response to LPS is unexpected, as other studies report that leukocyte responses to LPS can begin within 1–3 days and attenuate between 3–7 days after the challenge in reptiles (Merchant et al., 2006; Lind et al., 2020). Thus, it is possible that the majority of the leukocyte responses occurred and subsided between the baseline and 7-day sample. While we did see a significant increase in monocytes and several significant smooth terms for monocyte, lymphocyte, and heterophil ratios for both antigens, the relatively low leukocyte numbers and large variation between individuals limits the explanatory value of these results.

Overall, this study highlights the differences between two commonly used antigens in immune function research and characterizes the dynamics of the humoral immune response in an often-studied reptile. The more robust and uniform humoral response to KLH compared to LPS is likely due to the novelty of KLH and humoral responses to KLH requiring T cell help. This suggests that KLH is a more reliable antigen if looking specifically at the humoral immune system. Alternatively, while LPS failed to elicit IgY production in our study, it is known to produce a robust innate response characterized by inflammatory pathways and has led to IgY production in other reptile taxa, suggesting it can be an appropriate choice for humoral immune research and may have greater ecological relevance due to its ubiquity in nature (Mazgaeen and Gurung, 2020). However, the presence of LPS throughout the environment can lead to considerable inter-individual variance in the immune response based on an almost certain variation in the extent of previous exposure to LPS. KLH, while not an ecologically relevant antigen, provides the advantage of all individuals having no previous exposure to the antigen. We also observed sex-based differences in humoral immunity, males initially having a faster response. To our knowledge, this is only one of few studies in reptiles that has shown a more rapid humoral immune responses in males compared to females. Lastly, we observed increases in monocytes in KLH-injected animals shortly after injections, suggesting KLH contributes to the activation and mobilization of monocytes in reptiles as it does in mammals. Future work, however, should collect more extensive cell counts from blood smears to better characterize the leukocyte response to KLH.

To further characterize the differences in response to these two antigens, future work should assess the initial innate immune response, such as through the use of bacterial killing ability assays (BKA), which are commonly used to measure components on the innate immune system. Measurement of IgM antibodies can better characterize the early humoral response and can help determine when isotype switching occurs. Additionally, many extracellular pathogens contain a mixture of TI and TD antigens, meaning it is worthwhile to look at the humoral response to both types of antigens in the species being studied (Janeway et al., 2001). Regardless of the response to different antigens, the KLH component of this study demonstrates the presence of a strong, specific humoral immune response that was faster than has been considered the norm in reptiles (Zimmerman et al., 2010). This highlights the importance of the humoral immune system in reptiles and the need to account for its role when assessing overall immunity.