Experimental Acquisition, Maintenance, and Transmission of Methicillin-Resistant Staphylococcus aureus by the Common Bed Bug, Cimex lectularius.

Here, we fed bed bugs through a membrane contaminated with methicillin-resistant Staphylococcus aureus (MRSA) at a concentration naturally present on human skin. We then determined the amount of viable MRSA present on their surface and internally over a period of 7 days. We also determined whether bed bugs that fed through the contaminated membrane could transmit MRSA to an uncontaminated membrane when taking a second blood meal 7 days later. Bed bugs acquired MRSA both externally on the cuticle surface as well as internally when feeding. MRSA was found to persist for 7 days both externally and internally in some bed bugs. Furthermore, MRSA replicated internally but not externally. Most importantly, bed bugs were able to transmit MRSA to an uncontaminated membrane feeder in 2 of 3 trials. These findings provide the first experimental support for the hypothesis that bed bugs may contribute to the transmission of MRSA in some settings.

A metatranscriptomic evaluation of viruses in field-collected bed bugs.

Cimex lectularius, known as the common bed bug, is a widespread hematophagous human ectoparasite and urban pest that is not known to be a vector of any human infectious disease agents. However, few studies in the era of molecular biology have profiled the microorganisms harbored by field populations of bed bugs. The objective of this study was to examine the viruses present in a large sampling of common bed bugs and related bat bugs (Cimex pipistrelle). RNA sequencing was undertaken on an international sampling of > 500 field-collected bugs, and multiple workflows were used to assemble contigs and query these against reference nucleotide databases to identify viral genomes. Shuangao bed bug virus 2, an uncharacterized rhabdovirus previously discovered in Cimex hemipterus from China, was found in several bed bug pools from the USA and Europe, as well as in C. pipistrelle, suggesting that this virus is common among bed bug populations. In addition, Shuangao bed bug virus 1 was detected in a bed bug pool from China, and sequences matching Enterobacteria phage P7 were found in all bed bug pools, indicating the ubiquitous presence of phage-derived elements in the genome of the bed bug or its enterobacterial symbiont. However, viral diversity was low in bed bugs in our study, as no other viral genomes were detected with significant coverage. These results provide evidence against frequent virus infection in bed bugs. Nonetheless, our investigation had several important limitations, and additional studies should be conducted to better understand the prevalence and composition of viruses in bed bugs. Most notably, our study largely focused on insects from urban areas in industrialized nations, thus likely missing infrequent virus infections and those that could occur in rural or tropical environments or developing nations.

A Comparative Study of Body Lice and Bed Bugs Reveals Factors Potentially Involved in Differential Vector Competence for the Relapsing Fever Spirochete Borrelia recurrentis.

Borrelia recurrentis is the causative agent of louse-borne relapsing fever and the only Borrelia species transmitted by an insect rather than a tick vector. While bed bugs (Cimex lectularius L.) are not established vectors of any human pathogens, a recent study reported that they may be competent vectors of B. recurrentis. However, many aspects of infection and transmission remain unclear in this possible secondary vector. Here, we carried out several quantitative laboratory studies to gain a better understanding of the host suitability of bed bugs relative to the established body louse vector as well as the factors that may affect the ability of bed bugs to transmit the pathogen. We fed bed bugs B. recurrentis and estimated the level and duration of infection in the hemolymph using live imaging. We performed quantitative PCR (qPCR) to examine whole-body spirochete levels and the occurrence of vertical transmission to progeny. We also developed an assay to compare the amounts of force required to release infectious hemolymph from recently engorged bed bugs and body lice. Finally, we analyzed humoral antibacterial activity in the hemolymph, hemolymph pH, and hemocyte activity in both insect species. Our results confirm that within 24 h of ingestion, B. recurrentis can penetrate the midgut epithelium of bed bugs and enter the hemolymph, overcoming a major host barrier, as in body lice. Once in the hemolymph, spirochetes remain visible for at least 4 days. Moreover, we show that bed bugs are more physically susceptible to crushing than body lice, suggesting that crushing is a feasible route for the natural dissemination of B. recurrentis from the hemolymph of bed bugs, as for body lice. Nonetheless, our data also indicate that bed bugs are suboptimal hosts for B. recurrentis, as the bacterium does not appear to proliferate to high levels or stably colonize the hemolymph and exhibits pleomorphism in this environment. In particular, our data suggest that hemolymph pH and unique cellular immune responses, rather than humoral effectors, may be involved in limiting spirochete survival in bed bugs. Notably, we document the formation of extracellular DNA traps by bed bug hemocytes for the first time. For these reasons, while bed bugs may be capable of limited transmission given their ecology, vector competence is probably minimal relative to body lice. Additional mechanistic studies of human pathogen infection of bed bugs may provide much-needed insight into the biological factors that restrict their ability to act as vectors and may reveal novel mechanisms of immunity.

Indirect impact of COVID-19 on the incidence rates of vector-borne diseases in Mexico.

INTRODUCTION: SARS-CoV-2 infection spatial and temporal distribution overlaps with endemic areas of vector-borne diseases (VBD), whose surveillance in Mexico has substantially changed since the first COVID-19 confirmed case. OBJECTIVES: To estimate and compare the incidence rates of VBDs before and after the introduction of SARS-CoV-2 in Mexico. METHODS: Retrospective study of VBD cases from 2014 to 2021. The incidence rates of each VBD in the period before (2014-2019) and after (2020-2021) the introduction of SARS-CoV-2 in Mexico were calculated and compared. RESULTS: Before the introduction of SARS-CoV-2, the incidence rates of VBDs were high and after the introduction of coronavirus there was a decrease in epidemiological indices; however, there was only statistically significant difference in the incidence rate of malaria (p ≤ 0.05) and other rickettsiae (p ≤ 0.05). CONCLUSIONS: Some measures to reduce COVID-19 cases, such as social distancing, home confinement, reductions in public transport and working at home (home office), probably temporarily decreased the number of VBD cases; however, there may be a resurgence of VBDs in the near future.

INTRODUCCIÓN: La distribución espacial y temporal de la infección por SARS-CoV-2 sobrepasa las áreas endémicas de enfermedades transmitidas por vector (ETV), cuya vigilancia en México ha cambiado sustancialmente a partir del primer caso confirmado de COVID-19. OBJETIVOS: Estimar y comparar las tasas de incidencia de las ETV antes y después de la introducción del SARS-CoV-2 en México. MÉTODOS: Estudio retrospectivo de casos de ETV de 2014 a 2021. Las tasas de incidencia de cada ETV en el periodo previo (2014-2019) y posterior (2020-2021) a la introducción del SARS-CoV-2 en México fueron calculadas y comparadas. RESULTADOS: Antes de la introducción del SARS-CoV-2, las tasas de incidencia de las ETV fueron altas y posterior a la introducción del coronavirus hubo un descenso en los índices epidemiológicos; sin embargo, solo se identificó diferencia estadística significativa en la tasa de incidencia de la malaria (p ≤ 0.05) y otras rickettsias (p ≤ 0.05). CONCLUSIONES: Algunas medidas para reducir los casos de COVID-19, como el distanciamiento social, el confinamiento domiciliario, la reducción en el aforo en el transporte público y el trabajo en casa, probablemente contribuyeron a disminuir temporalmente el número de casos de las ETV; sin embargo, puede haber rebrote de las ETV en el futuro cercano.

Molecular analysis of the blood meals and bacterial communities of bed bugs (Cimex lectularius L.) to assess interactions with alternative hosts.

Common bed bugs (Cimex lectularius L.) are hematophagous pests present in urban environments across the globe. It is widely established that they have a strong host preference for humans. However, there are records of C. lectularius feeding upon a range of mammalian and avian hosts, including rodents, in the field. There is little information available about how frequently common bed bugs feed on alternative hosts in residential settings, but understanding this phenomenon has implications for both management of infestations and public health. Here, we examined cohorts of C. lectularius collected from 13 different dwellings in the state of New Jersey, USA, that were known to be simultaneously infested with house mice (Mus musculus domesticus). Host-specific quantitative polymerase chain reaction (qPCR) was used to determine if blood meals were taken from mice, while 16S rRNA gene amplicon sequencing was used to screen the bed bugs for the presence of zoonotic bacterial pathogens. We found no evidence that any of the bed bugs we collected fed on mice. Furthermore, the insects harbored depauperate bacterial communities that did not include known human pathogens. However, host-specific qPCR detected feline DNA in a pool of bed bugs from one dwelling, suggesting that interaction with domestic pets should be further investigated. Although sampling in this study was limited, the approach described herein will be useful for additional studies of the interactions between bed bugs and alternative blood meal hosts.

Insecticidal Activity of Doxycycline against the Common Bedbug.

There is an ongoing need for safe and effective anti-bedbug compounds. Here, we tested the toxicity of three antimicrobial agents against bedbugs when administered orally. We reveal that doxycycline has direct insecticidal activity at 250 µg/ml (0.025%) that is particularly strong against immature bedbugs and appears to be independent of antimicrobial activity. Future studies to determine the mechanisms behind this property could be useful for the development of orally active insecticides or anti-bedbug therapeutics.

Ex vivo characterization of the circulating hemocytes of bed bugs and their responses to bacterial exposure.

Bed bugs (Cimex spp.) are urban pests of global importance. Knowledge of the immune system of bed bugs has implications for understanding their susceptibility to biological control agents, their potential to transmit human pathogens, and the basic comparative immunology of insects. Nonetheless, the immunological repertoire of the family Cimicidae remains poorly characterized. Here, we use microscopy, flow cytometry, and RNA sequencing to provide a basal characterization of the circulating hemocytes of the common bed bug, Cimex lectularius. We also examine the responses of these specialized cells to E. coli exposure using the same techniques. Our results show that circulating hemocytes are comprised of at least four morphologically distinct cell types that are capable of phagocytosis, undergo degranulation, and exhibit additional markers of activation following stimulation, including size shift and DNA replication. Furthermore, transcriptomic profiling reveals expression of predicted Toll/IMD signaling pathway components, antimicrobial effectors and other potentially immunoresponsive genes in these cells. Together, our data demonstrate the conservation of several canonical cellular immune responses in the common bed bug and provide a foundation for additional mechanistic immunological studies with specific pathogens of interest.

Virulence of entomopathogenic bacteria in the bed bug, Cimex lectularius.

Due in part to the development of insecticide resistance, the common bed bug, Cimex lectularius, has overcome human intervention efforts to make a global resurgence. The failure of chemical pesticides has created a need for novel strategies to combat bed bugs. While a number of insect pests are susceptible to the use of entomopathogenic microbes or microbial-derived toxins, biological control methods have not been thoroughly explored in bed bugs. Here, we tested the virulence of three entomopathogenic bacterial species in C. lectularius to determine their potential for bed bug control. We examined bed bug survival after inoculation with live or heat-killed Serratia marcescens, Pseudomonas fluorescens, and Bacillus thuringiensis israelensis at varying temperatures. We also analyzed the viability and growth of the same bacteria in infected bed bugs. All three bacterial species were pathogenic to bed bugs. However, the effects of S. marcescens and P. fluorescens were temperature-dependent while the lethality of B. thuringiensis israelensis was not. In addition, bacterial virulence was partly dependent on the route of infection but was not strongly associated with proliferation. Thus, our results suggest multiple possible mechanisms of microbial pathogenicity in the bed bug and indicate that entomopathogenic bacteria, or products derived from them, may have useful applications for bed bug control.

Mechanisms of Horizontal Cell-to-Cell Transfer of Wolbachia spp. in Drosophila melanogaster.

Wolbachia is an intracellular endosymbiont present in most arthropod and filarial nematode species. Transmission between hosts is primarily vertical, taking place exclusively through the female germ line, although horizontal transmission has also been documented. The results of several studies indicate that Wolbachia spp. can undergo transfer between somatic and germ line cells during nematode development and in adult flies. However, the mechanisms underlying horizontal cell-to-cell transfer remain largely unexplored. Here, we establish a tractable system for probing horizontal transfer of Wolbachia cells between Drosophila melanogaster cells in culture using fluorescence in situ hybridization (FISH). First, we show that horizontal transfer is independent of cell-to-cell contact and can efficiently take place through the culture medium within hours. Further, we demonstrate that efficient transfer utilizes host cell phagocytic and clathrin/dynamin-dependent endocytic machinery. Lastly, we provide evidence that this process is conserved between species, showing that horizontal transfer from mosquito to Drosophila cells takes place in a similar fashion. Altogether, our results indicate that Wolbachia utilizes host internalization machinery during infection, and this mechanism is conserved across insect species.IMPORTANCE Our work has broad implications for the control and treatment of tropical diseases. Wolbachia can confer resistance against a variety of human pathogens in mosquito vectors. Elucidating the mechanisms of horizontal transfer will be useful for efforts to more efficiently infect nonnatural insect hosts with Wolbachia as a biological control agent. Further, as Wolbachia is essential for the survival of filarial nematodes, understanding horizontal transfer might provide new approaches to treating human infections by targeting Wolbachia Finally, this work provides a key first step toward the genetic manipulation of Wolbachia.

Two insulin-like peptides differentially regulate malaria parasite infection in the mosquito through effects on intermediary metabolism.

Insulin-like peptides (ILPs) play important roles in growth and metabolic homeostasis, but have also emerged as key regulators of stress responses and immunity in a variety of vertebrates and invertebrates. Furthermore, a growing literature suggests that insulin signaling-dependent metabolic provisioning can influence host responses to infection and affect infection outcomes. In line with these studies, we previously showed that knockdown of either of two closely related, infection-induced ILPs, ILP3 and ILP4, in the mosquito Anopheles stephensi decreased infection with the human malaria parasite Plasmodium falciparum through kinetically distinct effects on parasite death. However, the precise mechanisms by which ILP3 and ILP4 control the response to infection remained unknown. To address this knowledge gap, we used a complementary approach of direct ILP supplementation into the blood meal to further define ILP-specific effects on mosquito biology and parasite infection. Notably, we observed that feeding resulted in differential effects of ILP3 and ILP4 on blood-feeding behavior and P. falciparum development. These effects depended on ILP-specific regulation of intermediary metabolism in the mosquito midgut, suggesting a major contribution of ILP-dependent metabolic shifts to the regulation of infection resistance and parasite transmission. Accordingly, our data implicate endogenous ILP signaling in balancing intermediary metabolism for the host response to infection, affirming this emerging tenet in host-pathogen interactions with novel insights from a system of significant public health importance.

Human IGF1 regulates midgut oxidative stress and epithelial homeostasis to balance lifespan and Plasmodium falciparum resistance in Anopheles stephensi.

Insulin and insulin-like growth factor signaling (IIS) regulates cell death, repair, autophagy, and renewal in response to stress, damage, and pathogen challenge. Therefore, IIS is fundamental to lifespan and disease resistance. Previously, we showed that insulin-like growth factor 1 (IGF1) within a physiologically relevant range (0.013-0.13 µM) in human blood reduced development of the human parasite Plasmodium falciparum in the Indian malaria mosquito Anopheles stephensi. Low IGF1 (0.013 µM) induced FOXO and p70S6K activation in the midgut and extended mosquito lifespan, whereas high IGF1 (0.13 µM) did not. In this study the physiological effects of low and high IGF1 were examined in detail to infer mechanisms for their dichotomous effects on mosquito resistance and lifespan. Following ingestion, low IGF1 induced phosphorylation of midgut c-Jun-N-terminal kinase (JNK), a critical regulator of epithelial homeostasis, but high IGF1 did not. Low and high IGF1 induced midgut mitochondrial reactive oxygen species (ROS) synthesis and nitric oxide (NO) synthase gene expression, responses which were necessary and sufficient to mediate IGF1 inhibition of P. falciparum development. However, increased ROS and apoptosis-associated caspase-3 activity returned to baseline levels following low IGF1 treatment, but were sustained with high IGF1 treatment and accompanied by aberrant expression of biomarkers for mitophagy, stem cell division and proliferation. Low IGF1-induced ROS are likely moderated by JNK-induced epithelial cytoprotection as well as p70S6K-mediated growth and inhibition of apoptosis over the lifetime of A. stephensi to facilitate midgut homeostasis and enhanced survivorship. Hence, mitochondrial integrity and homeostasis in the midgut, a key signaling center for IIS, can be targeted to coordinately optimize mosquito fitness and anti-pathogen resistance for improved control strategies for malaria and other vector-borne diseases.

Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito host.

The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3-5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the midgut is necessary for the maintenance of midgut health as reflected in energy homeostasis and tissue repair and renewal.

Endosymbiont and gut bacterial communities of the brown-banded cockroach, Supella longipalpa.

The brown-banded cockroach (Supella longipalpa) is a widespread nuisance and public health pest. Like the German cockroach (Blattella germanica), this species is adapted to the indoor biome and completes the entirety of its life cycle in human-built structures. Recently, understanding the contributions of commensal and symbiotic microbes to the biology of cockroach pests, as well as the applications of targeting these microbes for pest control, have garnered significant scientific interest. However, relative to B. germanica, the biology of S. longipalpa, including its microbial associations, is understudied. Therefore, the goal of the present study was to quantitatively examine and characterize both the endosymbiont and gut bacterial communities of S. longipalpa for the first time. To do so, bacterial 16S rRNA gene amplicon sequencing was conducted on DNA extracts from whole adult females and males, early instar nymphs, and late instar nymphs. The results demonstrate that the gut microbiome is dominated by two genera of bacteria known to have beneficial probiotic effects in other organisms, namely Lactobacillus and Akkermansia. Furthermore, our data show a significant effect of nymphal development on diversity and variation in the gut microbiome. Lastly, we reveal significant negative correlations between the two intracellular endosymbionts, Blattabacterium and Wolbachia, as well as between Blattabacterium and the gut microbiome, suggesting that Blattabacterium endosymbionts could directly or indirectly influence the composition of other bacterial populations. These findings have implications for understanding the adaptation of S. longipalpa to the indoor biome, its divergence from other indoor cockroach pest species such as B. germanica, the development of novel control approaches that target the microbiome, and fundamental insect-microbe interactions more broadly.

Differences in Salmonella Typhimurium infection and excretion among laboratory and field strains of the German cockroach suggest a genomic basis for vector competence.

The German cockroach, Blattella germanica, can be a vector of human enteric bacterial pathogens, including Salmonella enterica serovar Typhimurium (S. Typhimurium). Transmission of such pathogens by cockroaches has largely been considered a passive mechanical process, but recent studies have argued against this dogma by demonstrating bacterial proliferation within the cockroach gut and the necessity of specific bacterial genes for successful transmission in the feces, revealing unappreciated biological complexity in the vector-pathogen relationship between cockroaches and S. Typhimurium. However, the influence of naturally occurring variation among cockroach populations on pathogen infection and dissemination has not been investigated. Thus, this study aimed to examine whether distinct strains of B. germanica exhibit differences in their ability to become infected by and disseminate S. Typhimurium. We performed controlled infections of one long-term laboratory strain and three recently field-collected strains reared under identical conditions, then compared bacterial loads in the body and excreta of individual insects. Separately, we also compared rates of necrophagy, a behavior known to contribute to the horizontal spread of S. Typhimurium among cockroaches. Our data show significant differences in infection susceptibility, pathogen shedding in the excreta, and necrophagy between laboratory and field strains as well as between some field strains. These observations represent the first evidence that genomic variation among cockroach populations may influence their ability to become infected by and disseminate pathogens, providing further support for the hypothesis that German cockroaches are active biological vectors rather than passive mechanical vectors of S. Typhimurium. Additional studies are needed to identify the genomic drivers of vector competence for S. Typhimurium in B. germanica.

Do bed bugs transmit human viruses, or do humans spread bed bugs and their viruses? A worldwide survey of the bed bug RNA virosphere.

BED BUGS: (Hemiptera: Cimicidae) are a globally distributed hematophagous pest that routinely feed on humans. Unlike many blood-sucking arthropods, they have never been linked to pathogen transmission in a natural setting, and despite increasing interest in their role as disease vectors, little is known about the viruses that bed bugs naturally harbor. Here, we present a global-scale survey of the bed bug RNA virosphere. We sequenced the metatranscriptomes of 22 individual bed bugs (Cimex lectularius and Cimex hemipterus) from 8 locations around the world. We detected sequences from two known bed bug viruses (Shuangao bedbug virus 1 and Shuangao bedbug virus 2) which extends their geographical range. We identified three novel bed bug virus sequences from a tenui-like virus (Bunyavirales), a toti-like virus (Ghabrivirales), and a luteo-like virus (Tolivirales). Interestingly, some of the bed bug viruses branch near to insect-transmitted plant-infecting viruses, opening questions regarding the evolution of plant virus infection. When we analyzed the viral sequences by their host's collection location, we found unexpected patterns of geographical diversity that may reflect humans' role in bed bug dispersal. Additionally, we investigated the effect that Wolbachia, the primary bed bug endosymbiont, may have on viral abundance and found that Wolbachia infection neither promotes nor inhibits viral infection. Finally, our results provide no evidence that bed bugs transmit any known human pathogenic viruses.

Horizontal transmission of Salmonella Typhimurium among German cockroaches and its possible mechanisms.

German cockroaches (Blattella germanica) can be both mechanical and biological (amplifying) vectors of enteric pathogens, including Salmonella enterica serovar Typhimurium (S. Typhimurium), which they acquire by feeding upon contaminated substances. Blattella germanica is also a gregarious species that shelters in groups and partakes in unique feeding behaviors such as conspecific coprophagy, necrophagy, and emetophagy. These properties create an interphase for potential horizontal transmission of pathogens among cockroach populations through the fecal-oral route, which could in turn enhance transmission to humans and other animals. Here, we performed a series of experiments to determine: (1) whether horizontal transmission of S. Typhimurium infection takes place in B. germanica, (2) the prevalence of the phenomenon, and (3) the route(s) through which it may occur. We reveal that true horizontal transmission of S. Typhimurium occurs among B. germanica. That is, uninfected cockroaches acquire infection of the gut when co-housed with orally infected conspecifics, albeit at low frequency. Furthermore, we provide definitive evidence that coprophagy and necrophagy are routes of transmission but could not exclude sharing of food or water as contributing routes. On the contrary, transmission by emetophagy appears less likely as oral regurgitates from infected cockroaches contained S. Typhimurium for less than one day after ingesting the bacteria. Together, our data enhance current understanding of the ecology of vector-borne S. Typhimurium transmission by cockroaches, implicating conspecific horizontal transmission as a phenomenon that contributes to maintaining infected cockroach populations independently of contact with primary sources of the pathogen. Although the relative importance of horizontal transmission of pathogens in cockroaches in the field remains to be determined, these results also highlight the important role that food and water sources in the local environment may play in cockroach-borne pathogen transmission and emphasize the importance of sanitation for not only abating infestations but also mitigating pathogen transmission.

The gut microbiota induces melanin deposits that act as substrates for fimA-mediated aggregation of Salmonella Typhimurium and enhance infection of the German cockroach vector.

When Salmonella Typhimurium is ingested by German cockroaches, the bacteria replicate in the gut and persist for at least 7 d, enabling transmission in the feces. However, the mechanisms that facilitate survival and persistence in the cockroach gut remain poorly detailed. We previously reported the formation of biofilm-like aggregate populations of S. Typhimurium in the gut of cockroaches upon ingestion. We also reported that deletion of the type-1 fimbrial subunit of S. Typhimurium, fimA, leads to a reduced bacterial load in the cockroach gut. Here, we link these observations and provide further insight into the mechanism and function of S. Typhimurium aggregation in the gut of the cockroach. We show that S. Typhimurium but not Escherichia coli forms aggregated populations in the cockroach gut, and that aggregate formation requires fimA but not the biofilm formation-related genes csgA and csgD. Furthermore, we show that S. Typhimurium aggregates are formed using small granular deposits present in the cockroach gut, which exhibit properties consistent with melanin, as substrates. These melanin deposits are prevalent in the guts of both immature and adult cockroaches from laboratory colonies and are correlated with increased gut bacterial density while being entirely absent in gnotobiotic cockroaches reared without exposure to environmental bacteria, indicating they are induced as a response to the gut microbiota. When cockroaches lacking melanin deposits in the gut are fed S. Typhimurium, they exhibit lower rates of infection than those harboring melanin deposits, demonstrating that microbiota-induced melanin deposits enhance infection of the gut of the vector. IMPORTANCE Cockroaches, including the German cockroach (Blattella germanica), can be both mechanical and biological vectors of pathogenic bacteria. Together, our data reveal a novel mechanism by which S. Typhimurium interacts with the cockroach gut and its microbiota that promotes infection of the vector. These findings exemplify the emerging but underappreciated complexity of the relationship between cockroaches and S. Typhimurium.

Effects of selected blood-derived factors on innate immunity in the human body louse.

BACKGROUND: The human body louse (Pediculus humanus humanus) is a host-specific hematophagous ectoparasite that frequently infests populations experiencing a breakdown of hygienic conditions. Body lice are also vectors for several bacterial human pathogens, including Bartonella quintana, the agent of trench fever. However, the factors that influence immunity and infection in body lice are poorly understood. Human infection with B. quintana is associated with alcoholism and homelessness and can coincide with elevated circulating levels of the cytokine IL-10 and the inflammatory marker neopterin. Hematophagous arthropods are capable of responding physiologically and immunologically to a variety of biomolecules present in the blood of their hosts. Therefore, we sought to investigate whether ingestion of alcohol, its metabolic product acetaldehyde, IL-10 or neopterin could affect innate immunity and infection in the body louse. METHODS: Groups of lice were provisioned multiple blood meals containing physiological concentrations of alcohol, acetaldehyde, IL-10 or neopterin, and expression of six previously identified immunity-related genes (Defensin 1, Defensin 2, Prophenoloxidase, Hemocytin, Noduler and Dual Oxidase) was examined by qRT-PCR. RESULTS: Alcohol, acetaldehyde and IL-10 had no significant effects on gene expression relative to blood-fed controls while ingestion of neopterin significantly downregulated expression of Defensin 1 and Defensin 2. Nonetheless, ingestion of neopterin concurrent with B. quintana had no significant effect on the load of infection, indicating that neopterin-induced repression of Defensin expression is insufficient to reduce resistance to the pathogen. CONCLUSIONS: Our findings demonstrate that the immune system of body lice can be affected by factors present in the blood of their human hosts and suggest potential conservation of the function of some immune molecules from human host to ectoparasite. Further, the discord between the effects of neopterin on immunity-related gene expression and B. quintana load highlights the complexity of the regulation of pathogen infection in the louse vector.

Survival of Salmonella Typhimurium in the hemolymph of the German cockroach vector is limited by both humoral immune factors and hemocytes but not by trehalose metabolism.

The German cockroach (Blattella germanica) has been linked to transmission of Salmonella enterica serovar Typhimurium (S. Typhimurium), but infection dynamics within this vector are poorly characterized. Our recent work has focused on S. Typhimurium infection in the cockroach gut. However, microbial dissemination to the hemolymph is an essential aspect of many vector-borne pathogen transmission cycles and could potentially contribute to S. Typhimurium colonization of cockroaches. Therefore, the goal of this study was to examine the ability of S. Typhimurium to disseminate, survive, and proliferate in the hemolymph of cockroaches after oral infection. We detected only low numbers of bacteria in the hemolymph of a minority of insects (~26%) after oral infection. Further, S. Typhimurium was unable to survive overnight in cell-free hemolymph. Several hypotheses to explain the inability of S. Typhimurium to colonize hemolymph were tested. First, we investigated the ability of S. Typhimurium to metabolize trehalose, the primary sugar in hemolymph. S. Typhimurium grew efficiently in vitro using trehalose as a sole carbon source and mutant strains lacking trehalose metabolism genes exhibited no growth deficiencies in media mimicking the composition of hemolymph, suggesting that trehalose metabolism ability is not a factor involved in restricting survival in hemolymph. On the other hand, heat-inactivated cell-free hemolymph was permissive of S. Typhimurium growth, demonstrating that survival in hemolymph is limited specifically by heat-labile humoral factors. The involvement of cellular immune responses was also investigated and cockroach hemocytes in culture were observed to internalize S. Typhimurium within 1 h of exposure. Most hemocytes harbored few to no bacteria after 24 h, indicating that hemocyte responses are additionally involved in clearing infection from the hemolymph. However, dense intracellular clusters of S. Typhimurium were observed sporadically, suggesting a small subset of hemocytes may serve as reservoirs for bacterial replication. Together, our results reveal that a minute proportion of ingested S. Typhimurium is able to escape the cockroach gut and enter the hemolymph, but this systemic population is limited by both humoral effectors and hemocytes. Thus, we conclude that invasion of the hemolymph appears minimally important for colonization of the cockroach vector and that colonization of the gut is the main driver of vector-borne transmission. Our insight into the antimicrobial mechanisms of cockroach hemolymph also highlights the strong ability of these prevalent pests/vectors to cope with frequent infectious challenges in septic habitats.

Effects of copper and zinc oxide nanoparticles on German cockroach development, indoxacarb resistance, and bacterial load.

BACKGROUND: The German cockroach, Blattella germanica, is a ubiquitous and medically significant urban pest. The ongoing development of insecticide resistance in global populations of B. germanica has complicated control efforts and created a need for improved tools. We previously reported that disruption of the gut microbiota by oral administration of the antimicrobial doxycycline reduced resistance in an indoxacarb resistant field strain and also delayed nymphal development and reduced adult fecundity. However, the application of doxycycline for cockroach control in the field is impractical. Here, we sought to determine whether two metal nanoparticles with known antimicrobial properties, copper (Cu) and zinc oxide (ZnO), have similar effects to doxycycline on the physiology of B. germanica and could provide more practical alternatives for control. RESULTS: We found that dietary exposure to 0.1% Cu nanoparticles, but not ZnO, significantly delays the development of nymphs into adults. However, neither of the nanoparticles altered the fecundity of females, and ZnO surprisingly increased resistance to indoxacarb in a resistant field strain, in contrast to doxycycline. Semi-quantitative polymerase chain reaction (qPCR) further revealed that prolonged dietary exposure (14 days) to Cu or ZnO nanoparticles at the low concentration readily consumed by cockroaches (0.1%) does not reduce the load of the bacterial microbiota, suggesting alternative mechanisms behind their observed effects. CONCLUSIONS: Together, our results indicate that ingestion of Cu nanoparticles can impact German cockroach development through an undetermined mechanism that does not involve reducing the overall load of the bacterial microbiota. Therefore, Cu may have some applications in cockroach control as a result of this activity but antagonistic effects on insecticide resistance should be considered when evaluating the potential of nanoparticles for cockroach control. © 2023 Society of Chemical Industry.