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Journal of Medical Entomology logoLink to Journal of Medical Entomology
. 2026 Jan 30;63(1):tjaf197. doi: 10.1093/jme/tjaf197

Cockroach gut microbiota is a significant source of endotoxin, a risk factor for asthma in cockroach-infested homes

Madhavi L Kakumanu 1,✉, Coby Schal 2
Editor: Michael Rust
PMCID: PMC12857570  PMID: 41615821

Abstract

The German cockroach, an obligate indoor pest, produces potent aeroallergens whose presence, along with endotoxins, are often reported as important indoor predictors of increased risk of morbidity in sensitized asthmatic children. In our recent analysis, we found significantly higher endotoxin concentrations in household dust from cockroach-infested homes than from uninfested homes in the same communities. We also found that both female and male cockroaches excreted large amounts of endotoxin in their feces. In this study, we hypothesized that if the cockroach gut microbiota is the major source of endotoxin, then all life stages would be expected to excrete endotoxin in relation to their gut microbial abundance. Using the kinetic Limulus amebocyte lysate assay, we found high levels of endotoxin in the feces of all life stages of the cockroach. In both laboratory-maintained and recently field-collected cockroaches, adult females produced 2.5- to 3-fold more endotoxin than males, consistent with their larger body mass and greater food consumption. Nymphs produced less endotoxin than adults, but the endotoxin concentration (endotoxin per mg) was higher in nymph feces than in adult feces. We found trace amounts of endotoxin in the feces of adults with axenic guts lacking microbiota. Lastly, endotoxin in fecal residues remained stable for at least 30 days at ambient room temperature. These results reveal that cockroaches expose sensitized people to a mix of allergens that are potent asthma triggers and endotoxins that can exacerbate the allergic response. Further research is warranted to understand their combined effects on asthma sensitization and exacerbation.

Keywords: Blattella, endotoxin, asthma, gut microbiota, gnotobiotic

Introduction

The German cockroach, Blattella germanica L. (Blattodea: Ectobiidae), is a synanthropic indoor pest of significant medical, veterinary, and economic importance (Lee et al. 2021, Schal and DeVries 2021). Infestations are found in residential settings, hospitals, farm buildings, and food preparation facilities and these cockroaches are known to carry enteric pathogens (Pai et al. 2004, Nasirian 2019, Schal and DeVries 2021). Moreover, they are a major source of indoor allergens that trigger allergic asthma in sensitized individuals, including children (Pomés and Schal 2020, Pomes and Arruda 2023). Disproportionately high levels of cockroach sensitization and childhood asthma were found in “inner-city” low-socioeconomic status (SES) urban neighborhoods, which often correlate with high levels of cockroach infestations, higher allergen levels, and other biological pollutants such as molds and endotoxins (Kang 1990, Rosenstreich et al. 1997, Rabito et al. 2011).

Endotoxins are the lipopolysaccharide components present in the outer cell membrane of Gram-negative bacteria. Exposure to airborne endotoxin has been shown to have varied effects in the development and exacerbation of asthma, depending on the age of exposure and the biotic and abiotic characteristics of the environment where exposure occurred. Exposure to endotoxins had a protective role against allergic asthma in children who grew up in farming communities (Nordgren and Bailey 2016, Stein et al. 2016). However, adults and children who grew up in urban neighborhoods and were exposed to high levels of endotoxins suffered increased risk of asthma morbidity (Wunschel and Poole 2016, Grant and Wood 2022). While co-occurrence of cockroaches and high levels of allergens and endotoxins have been reported in the homes of asthmatic children (Mendy et al. 2020), a direct demonstration that indoor pests serve as sources of clinically relevant amounts of endotoxin is lacking.

The German cockroach hosts a rich and diverse gut microbiome which is acquired through coprophagy and diet. It also harbors an obligatory endosymbiont, the Gram-negative Blattabacterium that resides in fat body cells throughout the hemocoel and in the ovaries, from which it is vertically transmitted in the eggs. A major proportion of the total microbiome comprises Gram-negative bacteria, which are also abundant in cockroach feces (Kakumanu et al. 2018). Lai (2017) assessed the levels of endotoxins in the feces of three cockroach species, including B. germanica, that were trapped in several homes in Hong Kong. The traps caught mostly adults, and their feces contained high amounts of endotoxin. However, the relative contributions of the broadly distributed endosymbiont and the gut to fecal endotoxin is unknown. Nonetheless, our recent in-home interventions conducted in Raleigh, NC, USA, revealed a positive correlation between cockroach infestation size and endotoxin concentration in settled floor dust (Kakumanu et al. 2026). Therefore, it is critical to understand the link between cockroaches and indoor endotoxins because severe and frequent German cockroach infestations are more common in urban low-SES homes, where cockroaches might be a source of a potent allergenic cocktail of allergens and endotoxins.

The German cockroach is an omnivore with a short generation time of five nymphal instars, adults live and reproduce for several months, and all life stages are found in infested homes. In this study, we aimed to determine the amount of fecal endotoxin produced and excreted into the indoor environment by all mobile life stages, including nymphs and adult males and females. We also compared these findings to feces obtained from three field-collected German cockroach populations. Moreover, since poor sanitation results in cockroach feces remaining in homes long after the cockroaches are eliminated, we investigated the stability of endotoxins in cockroach feces over a 30-day period. Lastly, to determine the source of fecal endotoxin, we generated gnotobiotic cockroaches that retained Blattabacterium but lacked a gut microbiome in their axenic gut.

Materials and Methods

Insect Collection, Rearing, and Feces Collection

We used four populations of the German cockroach. The Orlando Normal colony has been maintained in the laboratory since it was collected in 1947 in a Florida apartment. It is a common reference strain in insecticide resistance studies, and its genome has been sequenced and annotated (Harrison et al. 2018). In 2018, we collected cockroaches from three separate apartments in Raleigh, NC, USA and reared them for 6 years. The collection of cockroaches in homes was part of a larger study that was approved by the North Carolina State University Institutional Review Board (IRB #12188).

All cockroaches were reared at 27 °C and a photoperiod of L12: D12 h and provided with water and rodent diet (Purina 5001, PMI Nutrition International, St Louis, MO, USA). To synchronize the age of a large number of Orlando Normal nymphs needed for the experiments, we set up separate cages with 4- to 5-day-old adult females and 10-day-old males with food and water. Ootheca (egg case)-carrying females just before hatching of the eggs were moved to a new cage to monitor hatching. To synchronize the age of nymphs, 1- to 2-day-old first instars were moved to a new cage with food, shelter and water.

Feces were collected from first, third, and fifth (last) instar Orlando Normal nymphs at their active feeding stages (early- to mid-instar), and 4-day-old adult males and females of all four populations. Groups from each life stage (first instar: 100 nymphs; third instar: 50 nymphs; fifth instar: 25 nymphs; 10 adult males; 10 adult females) were placed in separate 10 × 90 sterile Petri plates and provisioned with water but no food to avoid contamination of feces by food particles. Feces were collected 24 h later. Each of these treatments was replicated 5 times. Results for the Orlando Normal (lab-colony) adult males and females were presented in Kakumanu et al. (2026) but are also shown here for easier comparison with nymphs and field-collected cockroaches.

Persistence of Endotoxin in Aged Feces

To examine if the endotoxin concentration in cockroach feces decreased over time after it was excreted, fresh feces were collected for 24 h from a large group (∼100 insects per each of three replications) of 1- to 7-day-old females and males, separately. A portion of each fecal sample was frozen immediately at baseline (0-day-old sample), and the remaining feces were kept at room temperature and sampled 15- and 30-day later. The samples were frozen immediately and stored at −80 °C before they were extracted and analyzed for endotoxins.

Feces from Gnotobiotic Cockroaches

We sought to understand the relative contribution of the gut microbiota and Blattabacterium to fecal endotoxin levels. To generate gnotobiotic cockroaches with an axenic gut, we sterilized the surface of egg cases and reared the hatching nymphs under sterile conditions. Normally, the gut of first instars is inoculated with microbes via coprophagy (Kopanic et al. 2001, Carrasco et al. 2014, Dominguez-Santos et al. 2024). Therefore, these procedures prevented the inoculation of the gut, and only vertically transmitted endosymbiotic microbes that reside in the fat body (e.g., Blattabacterium) were retained in the developing nymphs. Thus, we define the nymphs emerging from surface-sterilized egg cases as gnotobiotic because their gut was axenic (no microbiota, germ-free), but they retained Blattabacterium. B. germanica embryos that are close to hatching develop pigmented eyes and a prominent green spot, representing yolk in the hindgut. Egg case-carrying females with a “green line” in the egg case were briefly anesthetized with CO2 and each egg case was gently removed with forceps. Egg cases were surface sterilized by first washing them for 1 min in a 0.5% sodium hypochlorite solution, then 70% ethanol for 1 min, and three times with sterile water for 1 min each, following the procedures of Wada-Katsumata et al. (2015). To confirm the sterilization, the last wash (water) was plated on tryptic soy agar (TSA), incubated at 27 °C and monitored for 10 days. Further, 1 or 2 surface-sterilized egg cases were crushed in sterile 0.9% saline and plated for culturable microbes (Blattabacterium is not culturable). The surface washes and crushed homogenates of nonsterilized egg cases were similarly plated and incubated as controls.

We generated three treatment groups of newly hatched gnotobiotic first instars, one representing axenic conditions and two representing different levels of reinoculation with the feces of nonsterile adult females. One group of gnotobiotic nymphs was raised without adult females under sterile conditions, so they retained an axenic gut (sterile-no♀♀). The other two groups of gnotobiotic nymphs were reared with three nonsterile adult females for either 7 days (nonsterile-♀♀7 days), approximately the duration of the first stadium, or for approximately 30 days (nonsterile-♀♀30 days), nearly the full length of nymphal development. Each of the three treatments was replicated three times and each replicate had 30 nymphs. All the treatments were provided with autoclave-sterilized water and irradiated (sterilized) PicoLab® Laboratory Rodent Diet (5L0D, PMI Nutrition International, MN, USA). To minimize contamination during handling, the jars were not opened until the nymphs reached the adult stage, with the exception of one treatment (nonsterile-♀♀7 days), in which jars were opened within a biosafety cabinet only once after a week to remove females from the jars. We raised the nymphs to the adult stage, placed adult males and females in separate sterile Petri dishes for 24 h, and collected feces as in other assays. The feces were stored at −80 °C and analyzed for endotoxins.

Endotoxin Extraction and Analysis

The individual fecal samples were accurately weighed (Sartorius, Model 1712 MP8, Bohemia, NY, USA) and extracted with pyrogen-free water (PFW) with 0.05% Tween-20 at a concentration of 5 mg feces/ml, as described by Kakumanu et al. (2026). Endotoxin was extracted by gently shaking for 1 h at 22 °C on an orbital shaker at 200 rpm and the samples were centrifuged at 600 × g for 20 min at 4 °C. The supernatants were used for endotoxin quantification. The extracts from individual samples were analyzed at 4 dilutions with Lonza reagents using the Kinetic Chromogenic Limulus amebocyte lysate (kQLAL) assay (Lonza Bioscience, MD, USA), as described for the NHANES study (Thorne 2000, Thorne et al. 2005, Mendy et al. 2020). The increase in absorbance of samples was measured at 405 nm every 30 s over 90 min using a SpectroMax microplate reader and Softmax Pro 5.4 analysis software (Molecular Devices, Sunnyvale, CA, USA). The samples were evaluated against a 12-point standard curve using E. coli 055: B5 standard endotoxin (Lonza). Endotoxin concentrations in fecal samples are reported in Endotoxin Units (EU)/mg feces or EU/insect/day. The lower limit of detection in these assays was 0.005 EU/ml.

Data Analysis

Statistical analysis was performed using JMP 18.0 for Windows (SAS Institute, Cary, NC, USA) and Stata Release 9 (Stata Corp LB, College Station, TX, USA). Data are expressed as mean and standard error. The fecal and endotoxin data across life stages, persistence data, and gnotobiotic experiment data were analyzed using one-way ANOVA followed by Tukey’s HSD test at a significance level of α < 0.05. The field population data was analyzed using Welch’s t-test.

Results

Fecal Endotoxin across Different Life Stages

The amount of feces excreted per individual per day by Orlando Normal German cockroaches increased with life stage, with adults excreting significantly more feces than nymphs (Fig. 1A). Adult females produced significantly more feces per day than all other stages, including adult males, consistent with their higher food consumption. The amount of endotoxin excreted in feces followed a similar pattern, with adult females excreting the highest amounts of endotoxin (nearly 5,000 EU/day), 6.6-fold more than adult males (Fig. 1B). Analysis of the endotoxin concentration in feces revealed, however, that on a mass basis, the endotoxin concentration in fifth instar nymph feces was significantly higher than in adult female feces (Fig. 1C). The endotoxin concentrations in female and male feces were not significantly different, even though the concentration in females (2,900 EU/mg feces) was twice the concentration in male feces (1,400 EU/mg). Overall, each male and female excreted 750 and 5,000 EU per day in their feces, but the endotoxin concentration in feces was higher in immature stages than in adults, ranging from 3,200 to 5,700 EU/mg of feces.

Fig. 1.

Fig. 1.

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Endotoxin is excreted in the feces of all life-stages of the German cockroach. (A) Amount of feces (mg) produced by individual cockroaches per day. (B) Amount of endotoxin produced per cockroach per day. (C) Concentration of endotoxin detected per mg of cockroach feces. Each bar represents the mean + SEM of a different life-stage, and each treatment was replicated 5 times (n = 5). Stages and the number of insects per replication: N1 = 100 first instars, N3 = 50 third instars, N5 = 25 fifth (last) instars, 10 adult females, and 10 males (Totals: 500 first instars, 250 third instars, 125 fifth instars, 50 females, and 50 males). The P-value for one-way ANOVA is shown in each panel. Life-stages within each panel were compared with Tukey’s HSD test and means that do not share lowercase letters are significantly different. Results for the Orlando Normal (lab-colony) adult males and females were presented in (Kakumanu et al. 2026) but are also shown here for easier comparison with nymphs and field-collected cockroaches.

We conducted a similar analysis of fecal material collected from adult males and females from three populations collected in cockroach-infested homes. As in the lab colony (Orlando Normal), females from each of the three apartment populations excreted significantly more feces than males (Fig. 2A). Considering the means of all three apartments combined, females excreted 3.6-fold more feces than males (Welch’s t-test, t16.8 = 7.004, P < 0.0001). The amount of feces excreted by both females and males was significantly different across the three apartments and the Orlando Normal adults (Kruskal–Wallis test, Females: Chi-square(3) = 8.257, P = 0.041; Males: Chi-square(3) = 13.606, P = 0.0035). However, females from all three apartments excreted similar amounts of feces, and feces production in females from two apartments (1 and 3) and Orlando Normal females was not significantly different. Females from apartment 2 excreted significantly less feces than Orlando Normal females (Fig. 2A). Males from apartment 1 and Orlando Normal males excreted similar amounts of feces, and both groups were significantly different from males from apartments 2 and 3. Likewise, in all three apartment-collected populations, females produced significantly more endotoxin than males (Fig. 2B) and considering the means of all three apartments combined, females excreted 3.4-fold more endotoxin per day than males (Welch’s t-test, t15.6 = 4.586, P = 0.0003). However, the endotoxin concentrations in female and male feces were similar in all three apartments (Fig. 2C), including when considering the means of all three apartments combined (Welch’s t-test, t26.6 = 0.866, P = 0.394). Overall, despite high variation in the amounts of endotoxin excreted across the four populations, female cockroaches excreted significantly more endotoxin than males.

Fig. 2.

Fig. 2.

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Endotoxins excreted in the feces of adult males and females of three field-collected populations of the German cockroach. As in Fig. 1, (A) Amount of feces (mg) produced by individual cockroaches per day. (B) Amount of endotoxin produced per cockroach per day. (C) Concentration of endotoxin detected per mg of cockroach feces. Each bar represents the mean + SEM of males or females, and each treatment was replicated 5 times (n = 5). The number of adults per replication: 10 females and 10 males (Totals: 50 females and 50 males per population). Males and females across all three populations were compared within each panel with Welch’s t-tests. ns denotes P  ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Persistence of Fecal Endotoxin

We measured the endotoxin concentration in the feces of male and female cockroaches as the feces aged at ambient room conditions. Freshly collected female feces contained about 6,000 EU/mg (Fig. 3A), and although the endotoxin concentration declined with time, the day-30 concentration was not significantly different from the day-0 baseline concentration. Similarly, the endotoxin concentration in freshly collected male feces was about 4,000 EU/mg (Fig. 3B) and did not significantly change over time.

Fig. 3.

Fig. 3.

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Persistence of endotoxin (mean + SEM) in the feces of adult (A) males (B) females of the German cockroach over one month. Each treatment was replicated 3 times (n = 3). The number of adults per replication: 100 females and 100 males (Totals: 300 females and 300 males per treatment). The P-value for one-way ANOVA is shown in each panel. Endotoxin concentrations in female and male feces were compared separately over time with Tukey’s HSD test and means that do not share lowercase letters are significantly different.

Endotoxin from Gnotobiotic Cockroaches

No bacterial growth was observed on any of the TSA plates that were inoculated with the last surface wash of sterilized egg cases or with crushed egg cases that had been surface-sterilized (Fig. 4). In contrast, all plates inoculated with surface-washes or homogenized nonsterilized egg cases were positive for bacterial growth (Fig. 4). The gnotobiotic nymphs generated from the surface-sterilized egg cases were used for further testing.

Fig 4.

Fig 4.

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Confirmation of surface sterilization of egg cases by culturing. The extract (inoculum) was plated on Tryptic Soy agar (TSA) and incubated at 27 °C and monitored for 10 days. The images are of 3-day-old cultures. Control 1: One normal (nonsurface-sterilized) egg case was crushed in sterile 0.9% saline and a serial dilution of the homogenate was plated on TSA (n = 2). Control 2: The surface of five normal (nonsurface-sterilized) egg cases was washed with 0.9% saline for 1 min and the extract was plated (n = 2). After the surface-wash, one egg case per replication was crushed in sterile 0.9% saline and the homogenate plated on TSA (n = 2). Surface-sterilized: Five normal egg cases were surface-sterilized with 0.5% sodium hypochlorite solution, 70% ethanol and three washes with sterile water. The last water wash was plated for confirmation of sterility (n = 2). One surface-sterilized egg case per replication was crushed in sterile 0.9% saline and the homogenate was plated on TSA (n = 2). The absence of microbial growth in the surface-sterilized treatments confirmed that the egg cases were free of culturable microbes and appropriate for generating gnotobiotic nymphs. Microbial growth in the control treatments confirmed that the methodology was valid.

The amount of feces produced by male and female cockroaches raised from gnotobiotic nymphs with axenic guts (Fig. 5A) was similar to the amount excreted by Orlando Normal adults (Fig. 1A). Moreover, the absence of adult females (sterile-no♀♀; i.e., nymphs raised under sterile conditions) or the presence of nonsterile females for 7 days (nonsterile-♀♀7 days) or 30 days (nonsterile-♀♀30 days) during nymphal developmental did not significantly affect the amount of feces produced by the adult males and females that emerged from these nymphs (Fig. 5A). As in other assays, in all three treatments females produced significantly more feces than males. However, the amount and concentration of endotoxin in the feces were significantly different among the treatments. The feces of adults in the sterile-no♀♀ treatment, where gnotobiotic nymphs were reared without adults, had trace amounts of endotoxin (Fig. 5B), resulting in extremely low concentrations of endotoxin in feces (males: 6.8 EU/mg feces, Females: 8.6 EU/mg feces; Fig. 5C). In contrast, the feces of adult males and females from the nonsterile-♀♀7 days and nonsterile-♀♀30 days treatments, where gnotobiotic nymphs shared the cage with nonsterile adult females, exposing the nymphs to female feces, contained similar amounts of endotoxin as the feces of the respective Orlando Normal as well as field-collected cockroaches (Fig. 5B). As before, females excreted significantly more endotoxin than males. Thus, the endotoxin concentration in the feces of gnotobiotic cockroaches with non-axenic guts was significantly higher than in gnotobiotic cockroaches with axenic guts. Notably, both groups retained their fat body endosymbiotic community. However, within the same sex, little variation was found in the fecal endotoxin concentration in treatments nonsterile-♀♀7 days and nonsterile-♀♀30 days (Fig. 5C), indicating that early exposure to the feces of other cockroaches effectively reinoculated the gut of the gnotobiotic nymphs, which excreted endotoxin.

Fig. 5.

Fig. 5.

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Endotoxins excreted in the feces of adult male and female German cockroaches from three treatments using gnotobiotic first instars of the German cockroach. Gnotobiotic neonates (without a gut microbiome) were exposed to three treatments and reared to the adult stage: sterile-no♀♀, gnotobiotic nymphs reared under sterile conditions with no adults; nonsterile-♀♀7 days and nonsterile-♀♀30 days, gnotobiotic nymphs reared under nonsterile conditions with 3 normal (with gut microbiome) adult females for 7 days (duration of the first instar) or 30 days (duration of nymphal development), respectively. Feces were collected from the adults that emerged from these treatments. As in Fig. 1, (A) Amount of feces (mg) produced by individual cockroaches per day. (B) Amount of endotoxin produced per cockroach per day. (C) Concentration of endotoxin detected per mg of cockroach feces. Each bar represents the mean + SEM of males or females in each treatment, replicated 3 times (n = 3). The number of adults per replication: 10 females and 10 males (Totals: 30 females and 30 males per population). The P-value for one-way ANOVA is shown in each panel. Males and females across the three treatments were compared with Tukey’s HSD test and means within each panel that do not share lowercase letters are significantly different.

Discussion

The German cockroach, B. germanica, is a major pest of human-built structures and a clinically important source of indoor allergens, especially in low-SES households. As an extreme generalist omnivore, the German cockroach gut microbiome is highly diverse and dominated by Gram-negative bacteria (Carrasco et al. 2014, Pérez-Cobas et al. 2015, Kakumanu et al. 2018). Endotoxin, an important indoor pollutant, is a component of the outer cell wall membrane of Gram-negative bacteria, so we sought to understand the role of cockroaches in disseminating endotoxin in infested homes. Because cockroaches excrete several potent allergens in their feces (Gore and Schal 2005, Pomés and Schal 2020), as well as microbes with broad overlap with the gut microbiome (Kakumanu et al. 2018), we posited that co-excretion of allergens and endotoxins might result in co-exposure of residents to both, which is known to exacerbate asthma morbidity (Kulhankova et al. 2009). We sought to understand (a) the relative contribution of the gut microbiota and Blattabacterium to fecal endotoxin, (b) the comparative amounts of fecal endotoxin excreted by various life stages, and (c) the stability and persistence of fecal endotoxin.

Our results with gnotobiotic cockroaches with an axenic gut strongly support the hypothesis that the gut microbiota generates almost all the endotoxin in cockroach feces, while only trace amounts of fecal endotoxin could be attributed to the highly abundant Blattabacterium community in the fat body. The gut microbiome of the German cockroach is acquired primarily through coprophagy, diet and the environment (Kopanic et al. 2001, Carrasco et al. 2014, Pérez-Cobas et al. 2015, Kakumanu et al. 2018). In the sterile-no♀♀ treatment, gnotobiotic nymphs were raised in a sterile environment (sterilized food, water and cages) and without adults, which are the source of fecal microbes for coprophagy. These conditions prevented these nymphs from being inoculated with microbes. Therefore, they were devoid of a gut microbiome, reflected in the nearly undetectable levels of endotoxin in their feces. In the other two treatment groups, the gnotobiotic neonates shared cages with nonsterile females either during their first nymphal stadium or for their entire nymphal development. The feces of the adults that emerged from these treatments had high amounts of endotoxin, indicating that microbes were acquired by the gnotobiotic neonates through coprophagy and colonized their guts. Even though the adult females in the nonsterile-♀♀7 days treatment were removed after a week, the endotoxin amount and concentration in the feces of the emerging adults were as high as in normal females, indicating that inoculation of neonates in the first instar is sufficient for the nymphs to acquire and maintain a gut microbiota, consistent with the development and succession of the gut microbiome observed in the German cockroach (Carrasco et al. 2014).

Our previous microbiome analysis of whole body homogenates showed that Blattabacterium is highly abundant, representing approximately 50–70% of the 16S rRNA reads in both lab and field-collected males and females (Kakumanu et al. 2018). In contrast, Blattabacterium represented <0.5% of the 16S rRNA reads in cockroach feces. When nymphs were devoid of gut bacteria but retained Blattabacterium (treatment sterile-no♀♀), the total amount of endotoxin detected in their adult feces was <10 EU/mg feces compared to thousands of EU/mg feces when the gut microbiome was intact. Thus, the contribution of Blattabacterium to fecal endotoxin appears to be negligible compared to the contribution of the gut microbiota. Even so, Blattabacterium might represent a significant reservoir of nonfecal endotoxin. Since endotoxins are released at higher rates during bacterial cell lysis than from live bacteria (Sheehan et al. 2022), it is plausible that as cockroaches die and their bodies pulverize and become part of household dust, Blattabacterium-associated endotoxin might also be disseminated throughout the home. Interestingly, we previously reported that Wolbachia, an endosymbiont that resides in the hemocoel of the bed bug, was highly represented in 16S rRNA reads from household dust in bed bug-infested homes (Kakumanu et al. 2020); however, the contribution of Wolbachia-associated endotoxin in house dust was not measured. Accordingly, the contribution of Blattabacterium to the endotoxin load in infested homes remains unknown, but its high abundance in cockroach bodies suggests that dead cockroaches might contribute to even higher endotoxin loads than through cockroach feces alone.

We found that endotoxin is produced and excreted in the feces of all life stages of the German cockroach. The amount of feces produced by cockroaches per day increased with nymphal age and therefore with the size of nymphs, as expected based on the fact that larger nymphs eat more (Demark and Bennett 1994) and therefore defecate more. Similarly, we also found that the endotoxin concentration (units per mg) in the feces increased from the first to fifth (last) instar. This is likely related to the development and proliferation of their gut microbiota with age, where first instars have relatively low abundance of gut bacteria and the microbial diversity and abundance increase as nymphal development progresses (Carrasco et al. 2014). Thus, concomitant with nymphal development, endotoxin content increases in feces because (a) more feces are produced (mg per day) in concert with greater food consumption, (b) the gut bacterial load increases from the first instar to the adult stage, and (c) the representation of Gram-negative bacteria in the gut likely increases during nymphal development.

Among adults, 4-day-old females produced 3.3-fold more feces per day than males and their endotoxin concentration was 6.6-fold higher than in males. This is consistent with the females’ high food consumption that supports vitellogenesis and oocyte maturation (Hamilton and Schal 1988). A similar relationship was reported in the production of the cockroach allergen Bla g 1, which is also abundant in the feces (Fan et al. 2005, Gore and Schal 2005). Interestingly, although cockroach males produce less feces, and thus, less endotoxin per day, their overall fecal production and excretion of endotoxin might be closer to the females’. The female’s food intake is modulated by the reproductive cycle, which includes ∼7 days of intensive eating (and fecal excretion) in support of vitellogenesis, oocyte maturation and egg case production. In this study, we used 4-day old females that were within this active vitellogenic phase of the gonadotrophic cycle. However, this phase is followed by a protracted 21-day gestation period of minimal food intake and minimal excretion of feces; during her 28-day cycle each female eats ∼87 mg of food (Hamilton and Schal 1988). In contrast, males have a more consistent and low daily food intake averaging 1.6 mg per day, or ∼45 mg over 28 days (Schal and Wada-Katsumata 2021). Therefore, the overall contribution of adult males to the endotoxin load in infested homes might be underestimated in our study. Moreover, since adult males tend to forage more broadly than females, and especially in comparison to gravid females (Silverman 1986), males also might spread feces, containing allergens and endotoxin, more extensively throughout the home.

We detected high levels of endotoxin in the feces of male and female cockroaches collected in infested homes. As in lab colony adults, females produced more feces and endotoxin than male cockroaches. Moreover, the amounts of endotoxin released by both lab and field cockroaches were similar to the endotoxin levels reported from field-collected German cockroaches in Hong Kong (Lai 2017). Nevertheless, we found significant differences in both the total feces and endotoxin produced per day among the three home-collected populations, despite being reared on the same diet as the Orlando Normal lab colony for 6 years. Differences in feeding habits (McPherson et al. 2021) and the total gut bacterial loads among these populations might drive the variation in endotoxin content. This is consistent with our observations of relatively higher variation in the cockroach gut microbial diversity among apartments than in the lab population (Kakumanu et al. 2018). Similarly, significantly higher endotoxin levels (10-fold) were reported in wild Periplaneta americana L. (American cockroach) (Blattodea: Blattidae) than in a lab colony (Tungtrongchitr et al. 2012).

To mitigate asthma severity, a critical target in environmental interventions is the cockroach population, the source of allergens that trigger asthma. Successful interventions have reported significant reductions in both cockroaches and allergens (Arbes et al. 2003, 2004, Sever et al. 2007, Wang and Bennett 2009, Rabito et al. 2017). In a recent in-home environmental study, we detected high levels of endotoxin in cockroach infested homes, along with allergens. We discovered that significant reduction or elimination of the cockroach population also reduced the endotoxin load in homes (Kakumanu et al. 2026). Nevertheless, reservoirs of dead cockroaches, their feces, allergens, and endotoxins remained in the homes. Therefore, we examined the stability and persistence of endotoxins in cockroach feces. Our results showed little change in the endotoxin concentration after one month. These findings suggest that even though we find an overall reduction in the amounts of allergens and endotoxins after eliminating the cockroaches, thorough cleaning is needed to remove both allergens and endotoxins, which are persistent and might become part of household dust, air-borne, and thus, inhalable and adsorbed to various surfaces. Endotoxin is highly heat stable and hydrophobic (Gorbet and Sefton 2005), so a more thorough cleaning with appropriate solvents is essential once their source—the cockroach—is eliminated.

Contrasting effects have been proposed for endotoxin in pulmonary disease. While protective effects have been documented among farm children, exposure to high levels of endotoxins in nonfarming communities elicits inflammatory and allergic responses (Thorn 2001, Liu 2002, Thorne et al. 2005). Conversely, the role of cockroaches as producers of potent allergens that trigger allergic and asthmatic responses is incontrovertible. Thus, co-exposure to endotoxin and allergens might exacerbate the immune response to a higher degree than in response to each alone (Williams et al. 2005). For example, repeated co-exposures of neonatal and juvenile mice with endotoxin and cockroach allergen significantly increased lung inflammation, serum IgE and IgG(1), and alveolar wall thickness, and decreased airspace volume density. More importantly, the responses to co-exposures were more than additive, compared to separate exposures to allergens and endotoxins (Kulhankova et al. 2009).

Another important consideration is the use of cockroach extracts in diagnosis and immunotherapy of allergic diseases. Recent studies have shown that commercial extracts used in immunotherapy are highly variable, in part because they use extracts of different sources and different life stages of the cockroach (Birrueta et al. 2019, Mindaye et al. 2020). We now add the caveat that unless the extracted allergens are carefully purified, such extracts may contain variable concentrations of endotoxin, which alone might stimulate an allergic response, and might do so in concert with cockroach allergens. Indeed, whole body cockroach extracts licensed for clinical use in humans contained high levels of endotoxin ranging from 1,617 to 13,418 EU/ml (Birrueta et al. 2019, Glesner et al. 2019). Interestingly, cockroach feces extracts used for research contained up to 342,622 EU/ml, again highlighting the abundance of endotoxins in cockroach feces.

Our study has several limitations. The first relates to the representation of home-collected cockroaches. Only three apartments were represented, and in all three cases, cockroaches were reared in the lab for several years. Therefore, it would be worthwhile to collect fresh cockroaches from more apartments and quantify endotoxin associated with their feces immediately after collection. Second, our endotoxin-stability assays were limited to one month at ambient temperature. Studies are needed to extend this time-course and introduce environmental variation, such as higher temperature and humidity. Also, the effects of various cleaning agents on endotoxin need to be explored. Finally, clinically relevant endotoxin levels are measured in air (EU per m3), so the relationship between endotoxin excretion by cockroaches and air-borne endotoxin needs to be investigated. Notably, we detected low levels of endotoxin (∼10 EU/mg) in the dust trapped on heating, ventilation and air conditioning (HVAC) filters (Kakumanu et al. 2026), suggesting that it becomes air-borne, likely in association with small dust particles.

In summary, this study shows for the first time that high amounts of endotoxin are excreted by all life stages of the German cockroach as well as by cockroaches collected in infested homes. Our findings indicate that endotoxin is stable and might be highly persistent in the home environment and therefore warrant thorough cleaning following cockroach elimination to eliminate dead cockroaches, allergens and endotoxins. Large cockroach infestations in the indoor home environment produce a poorly characterized mixture of highly abundant potent pollutants—including allergens and endotoxins—that can sensitize atopic children and trigger adverse health outcomes. Thus, further studies are warranted to understand the comprehensive and combinatorial effects of cockroach-associated pollutants on pulmonary health.

Acknowledgements

We are grateful to residents in Raleigh North Carolina for their participation in this and related study, which allowed us to sample cockroaches in their homes.

Contributor Information

Madhavi L Kakumanu, Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC, USA.

Coby Schal, Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC, USA.

Funding

The research was supported in part by a grant from the US Department of Housing and Urban Development Healthy Homes program (NCHHU0081-24), a Pilot Project awarded by the Center for Human Health and the Environment under P30ES025128 from the National Institute of Environmental Health Sciences, the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (1R21AI187857-01), Research Capacity Fund (HATCH) (project NC02639) from the U.S. Department of Agriculture National Institute of Food and Agriculture, the Pest Management Foundation, and the Blanton J. Whitmire Endowment at North Carolina State University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors.

Conflicts of Interest

None declared.

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