Historical and Contemporary Control Options Against Bed Bugs, Cimex spp.

Bed bugs (Hemiptera: Cimicidae) are an important group of obligate hematophagous urban insect pests. The global resurgence of bed bugs, involving the common bed bug, Cimex lectularius L., and the tropical bed bug, Cimex hemipterus (F.), over the past two decades is believed to be primarily due to the development of insecticide resistance, along with global travel and poor pest management, which have contributed to their spread. This review examines and synthesizes the literature on bed bug origins and their global spread and the literature on historical and contemporary control options. This includes bed bug prevention, detection and monitoring, nonchemical and chemical control methodologies (and their limitations), and potential future control options. Future research needs are highlighted, especially the factors behind the modern resurgence, the necessity of identifying differences between the two bed bug species relevant to control, and the need to improve insecticide test protocols and management strategies.

Geographical patterns and mechanisms of Cimex lectularius Linnaeus, 1758, and Cimex hemipterus Fabricius, 1803 (Hemiptera: Cimicidae) resistance to insecticides: a systematic review and meta-analysis.

This study aimed to review published scientific literature on bed bugs in countries where insecticide resistance has been reported worldwide from 2000 to 2021. Electronic databases, including Scopus, PubMed, and WOS, were searched. Out of 606 articles found in the initial search, we selected 57 articles, of which 40 articles had reported on Cimex lectularius (C. lectularius), and 22 papers had reported on Cimex hemipterus (C. hemipterus). Most studies on insecticide resistance were carried out on C. lectularius in North America (14, 35%) and C. hemipterus in Asia (16, 72.7%). The most common method used to detect bed bug resistance to insecticides was toxicological bioassay with an overall random pooled effect size of 0.38 (95% CI: 0.23-0.53) in C. lectularius and 0.46 (95% CI: 0.27-0.65) in C. hemipterus. Resistance to pyrethroids was reported against C. lectularius with an overall pooled effect size of 0.75 (95% CI: 0.56-0.94) and C. hemipterus with an overall pooled effect size of 0.81 (95% CI: 0.57-0.93) in 33.40 (82.5%) and 19.22 (86.3%) published articles, respectively. A very high resistance level to pyrethroids in both studied species was observed, and resistance ratios at the highest level were 76389.3 and 315.5 in C. lectularius and C. hemipterus, respectively. Resistance mechanisms against pyrethroids were reported from most locations except Iran and Thailand, but these mechanisms were not studied in other insecticide groups. These reports indicate that chemical control options for bed bugs are limited. Therefore, a combination of chemical and non-chemical strategies is recommended for bed bug control.

An Earliest Endosymbiont, Wolbachia massiliensis sp. nov., Strain PL13 from the Bed Bug (Cimex hemipterus), Type Strain of a New Supergroup T.

The symbiotic Wolbachia are the most sophisticated mutualistic bacterium among all insect-associated microbiota. Wolbachia-insect relationship fluctuates from the simple facultative/parasitic to an obligate nutritional-mutualistic association as it was the case of the bedbug-Wolbachia from Cimexlectularius. Understanding this association may help in the control of associated arthropods. Genomic data have proven to be reliable tools in resolving some aspects of these symbiotic associations. Although, Wolbachia appear to be fastidious or uncultivated bacteria which strongly limited their study. Here we proposed Drosophila S2 cell line for the isolation and culture model to study Wolbachia strains. We therefore isolated and characterized a novel Wolbachia strain associated with the bedbug Cimexhemipterus, designated as wChem strain PL13, and proposed Wolbachiamassiliensis sp. nov. strain wChem-PL13 a type strain of this new species from new supergroup T. Phylogenetically, T-supergroup was close to F and S-supergroups from insects and D-supergroup from filarial nematodes. We determined the 1,291,339-bp genome of wChem-PL13, which was the smallest insect-associated Wolbachia genomes. Overall, the wChem genome shared 50% of protein coding genes with the other insect-associated facultative Wolbachia strains. These findings highlight the diversity of Wolbachia genotypes as well as the Wolbachia-host relationship among Cimicinae subfamily. The wChem provides folate and riboflavin vitamins on which the host depends, while the bacteria had a limited translation mechanism suggesting its strong dependence to its hosts. However, the clear-cut distinction between mutualism and parasitism of the wChem in C. hemipterus cannot be yet ruled out.

Isolation and characterization of human DNA recovered from Cimex hemipterus (F.) (Hemiptera:Cimicidae).

The ability to isolate and generate a DNA profile from human DNA recovered from tropical bed bugs (Cimex hemipterus) for identifying individuals can be useful for public health, forensic, and medical entomology. In this study, genomic DNA was recovered from both male and female bed bugs at every time interval tested (0, 1, 3, 5, 7, 14, 30, and 45 days post blood meal). The total DNA concentrations recovered from male bed bugs ranged from 12.93 to 65.97 ng/µL, while the total DNA concentrations from female bed bugs ranged from 8.93 to 44.53 ng/µL. However, based on the results from the BLAST search and PCR products, human DNA could be detected from female bed bugs at 0, 3, 5, 14, and 30 days post blood meal using the D18S51 marker. Concentrations of PCR products of the D18S51 locus from male bed bugs ranged from 4.20 to 35.50 ng/µL, whereas, for female bed bugs, concentrations ranged from 4.31 to 22.47 ng/µL. These were generally higher compared to the PCR products of the first hypervariable part (HVR1) marker. The results indicate the HVR1 locus was less sensitive than the D18S51 locus.

Efficacy of steam and diatomaceous earth dust against the tropical bed bug Cimex hemipterus (F.) under laboratory and field conditions.

BACKGROUND: Over the past two decades, bed bugs (Cimex spp.) have resurged as common urban pests around the world. The search for efficient and safe control measures has become a key interest among researchers, manufacturers, and pest control professionals. In this study, we evaluated and compared the efficacy of steam, diatomaceous earth (DE) dust, and a combination of both against tropical bed bugs (Cimex hemipterus (F.)) under laboratory and field conditions. RESULTS: In the laboratory study, the mortality of bed bugs after 2 days of exposure to DE dust was 100%. When bed bugs stayed on the surface of an object or in cracks, a brief steam treatment (1 s) caused 100% mortality. However, when bed bugs were hidden under a fabric cover, steam application for 10 s only caused 89 ± 6% mortality. Bed bugs that survived steam treatment exhibited reduced feeding activity. In a 14-week long study, there was no significant difference in the reduction rate of bed bugs between steam treatment and DE dust treatment. A 37-week long control study showed that steam and steam plus DE dust treatments eliminated 97-100% of the infestations. CONCLUSION: Applying steam and DE dust are effective strategies for eliminating natural tropical bed bug infestations. Continuous follow-up monitoring and treatment until no bed bugs are found are crucial in completely eliminating the infestation of tropical bed bugs. © 2024 Society of Chemical Industry.

Repellent effect of two anthranilates against the tropical bed bug Cimex hemipterus (Hemiptera: Cimicidae) demonstrated in laboratory assays.

BACKGROUND: The bites and blood sucking of bed bugs (Cimex spp.) (Hemiptera: Cimicidae) pose a serious threat to human physical and mental health. Application of an effective repellent can prevent or reduce bed bug bites. Previous studies on repellent screening mainly focused on Cimex lectularius L. In this study, we investigated the repellent effect of two safe food additives, ethyl anthranilate (EA) and butyl anthranilate (BA), against Cimex hemipterus (F.), and also explored the role of antennae and mouthparts on C. hemipterus perception of repellents. RESULTS: Both EA and BA had a strong repellent effect against tropical bed bugs and their repellency was similar or lower than that of N,N-diethyl-3-methyl benzoyl amide, depending on whether or not a CO2 source was present. EA had higher repellency than BA and exhibited repellency to C. hemipterus for 3 days when a CO2 source was present. C. hemipterus avoided resting on 20% EA- and BA-treated harborages. Applying 20% EA and BA on rabbit skin significantly reduced the blood intake of C. hemipterus within 2 h. C. hemipterus could perceive EA and BA after their antennae or mouthparts or both antennae and mouthparts were removed. CONCLUSION: Both EA and BA had strong repellency against C. hemipterus, with EA being more repellent. Ablation of antennae and mouthparts did not affect the perception of EA and BA by C. hemipterus. © 2023 Society of Chemical Industry.

Morphological, molecular, and MALDI-TOF MS identification of bed bugs and associated Wolbachia species from Cameroon.

After vanishing from the public eye for more than 50 years, bed bugs have resurged to become one of the most widely discussed and heavily researched insect pests in the world. This study presents the basic information of infestations of tropical bed bugs, Cimex hemipterus (Hemiptera: Cimicidae), in Cameroon. A total of 248 immature stage and adult bed bug specimens were collected from households and a travel agency in Yaoundé and Douala, Cameroon. The ability of MALDI-TOF MS to identify bed bugs was tested using heads for adults and cephalothoraxes for immature stages. Microorganism screening was performed by qPCR and confirmed by regular PCR and sequencing. Based on morphometrical criteria, four stages of immature bed bugs are represented. Of the 248 bed bug specimens morphologically identified as Cimex hemipterus, 246 (77 males, 65 females and 104 immature specimens) were submitted to MALDI-TOF MS analysis. Of the 222 adults and immature specimens tested, 122 (59.9 %) produced good quality MALDI-TOF MS spectra (35 adults and 87 immature specimens). Blind testing allowed species level identification of 98.21 % of adult and immature C. hemipterus. Among the bacteria tested, only Wolbachia DNA was found in 12/246 (4.8 %) bed bugs. More surveys in the country are warranted to assess the true level of bed bug infestations, in order to take appropriate action for their control.

Protocol for tropical bed bug use as forensic tools.

As blood-feeding insects that feed on human hosts, bed bugs could be used in forensic investigations if they are present at a crime scene with no apparent evidence. This study describes how tropical bed bugs (Cimex hemipterus) can be used as forensic tools to collect valid human DNA samples. Short Tandem Repeat (STR) analysis was performed on collected bed bug samples, whereby the results indicate that the obtained quantities of human DNA are mostly substantial to facilitate a comprehensive genetic profiling process.

Species identification and pyrethroid resistance genotyping of recently resurgent Cimex lectularius and Cimex hemipterus in Korea.

The global resurgence of bed bug infestations, exacerbated by increasing international travel, trade, and insecticide resistance, has significantly impacted Korea. This study identified the bed bug species and performed pyrethroid resistance genotyping of recently resurgent bed bugs in Korea. Thirty-one regional bed bug samples were collected from 5 administrative regions: Gyeonggi-do (n=14), Seoul (n=13), Busan (n=2), Jeonllanam-do (n=1), and Chungcheongbuk-do (n=1). The samples underwent morphological and molecular identification. Twenty-four regional samples (77.4%) were identified as the tropical bed bug, Cimex hemipterus, and the remaining 7 regional samples (22.6%) were identified as the common bed bug, Cimex lectularius. The C. hemipterus regional samples carried at least three mutations associated with knockdown resistance (kdr), including 2 super-kdr mutations. The 7 C. lectularius regional samples possessed at least one of the 3 kdr-related mutations associated with pyrethroid resistance. This study confirms that the prevalent bed bug species recently in Korea is C. hemipterus, replacing the previously endemic C. lectularius. Additionally, the rise in bed bug populations with pyrethroid resistance underscores the necessity of introducing alternative insecticides.

Behavioural Responses of Tropical Bed Bug Cimex hemipterus (F.) (Hemiptera: Cimicidae) to Coloured Harbourage.

Population of the tropical bed bug Cimex hemipterus (F.) (Hemiptera: Cimicidae), a temporary ectoparasite on both humans and animals, have surged in many tropical countries. Tropical bed bugs preferences when selecting a suitable harbourage and oviposition site were investigated. Two-choice and three choice colour assays were conducted to determine whether bed bugs will choose black, red or white coloured harbourages. Then, 50 1st instar were reared in containers containing black, red and white (control) paper served as the harbourages and observed for 12 weeks. Both fed and starve male, female and nymph strongly preferred red and black coloured harbourage compared to white coloured harbourage. Oviposition assays showed that female bed bugs prefered to laid their eggs on red coloured harbourages compared to black coloured harbourages. Rearing experiment showed that there was no significant difference (p > 0.05) between final population size of tropical bed bug. However, tropical bed bugs reared in container with red paper (600 ± 89.238) have the highest number of individuals followed by black (473 ± 133.841) and white (485 ± 84.234) paper. Bed bug preference towards coloured harbourage provide useful information for those developing new bed bug control method or improving bed bug infestation monitoring devices.

A Case Study on Tropical Bed Bug, Cimex hemipterus (Hemiptera: Cimicidae) Infestation and Management in Dormitories.

Numerous bed bug research papers have been published in the past 20 yr as a result of bed bug (Cimex spp.) (Hemiptera: Cimicidae) resurgence in the world. Yet, few of them focused on the management of the tropical bed bug, C. hemipterus (F.). Here, we describe a case of tropical bed bug infestation in two dormitory buildings and effectiveness of a tropical bed bug treatment program. The study site consisted of 125 dormitories in two buildings. An initial building-wide monitoring with ClimbUp interceptors revealed 25 infestations. The spatial distribution of bed bug infested rooms showed a significant aggregated distribution pattern with same infestation status for neighboring units sharing walls. All infested rooms were monitored every 2 wk and treated using a combination of steam and diatomaceous earth (DE) dust application if bed bugs were still found. For the 25 initially identified infested rooms, after 14 wk treatment, 44% of them no longer had bed bugs, and the mean number of bed bugs captured per room decreased by 94.1%. A combination of steam and DE dust treatment is an effective strategy for suppressing tropical bed bug infestations in dormitory environment.

Perspective on Biology and Management of Bed Bugs: Introduction.

Bed bugs are an important group of medical and urban insect pests. They are obligate blood-feeders. Their bites may cause skin irritation and allergic reactions and, under some circumstances, may lead to mental and other health issues. Despite numerous discoveries on the biology of these obnoxious pests and progress in control strategies over the last two decades, bed bugs continue to preferentially plague those from low socioeconomic communities because the poor generally could not afford effective control options. As a result, such infestations in poorer communities serve as a reservoir for wider society. This Special Collection of the Perspective on Biology and Management of Bed Bugs presents nine original research papers on bed bug detection, insecticide performance and resistance, nonchemical treatment, fungal biopesticides, and pest management procurement and contracts. We hope that these investigative findings will spur research on safer, more affordable, and effective control options in the future.

The Efficacy of a Pyrethroid-impregnated Mattress Liner on Multiple International Strains of Cimex lectularius (Hemiptera: Cimicidae) and Cimex hemipterus (Hemiptera: Cimicidae).

Modern bed bugs are resistant to multiple insecticide classes, particularly the pyrethroids. The efficacy of pyrethroid-impregnated mattress liners marketed for bed bug management has been variable. This study evaluated the efficacy of a permethrin-impregnated mattress liner, ActiveGuard, against 24 bed bug strains, consisting of both Cimex hemipterus (F.) and Cimex lectularius L. A 'mat assay', employing an allethrin-impregnated mat, was used to establish the pyrethroid resistance profile of all strains. Three experiments were conducted to evaluate the effect of ActiveGuard exposure on bed bug knockdown: 1) exposing the bed bugs continuously on the liner for up to 24 d, 2) holding the bed bugs on the liner for either 4 or 6 h, and 3) placing a noninsecticide treated fabric above the liner with the bed bugs held continuously on top. Our results indicated that all modern strains (collected within the last 15 years during the current resurgence) were pyrethroid-resistant, although the magnitude of resistance was highly variable between strains. In the continuous exposure study, an incomplete knockdown was recorded for most modern bed bug strains, with some having no knockdown even up to 7 d of constant exposure. In the 4 or 6 h exposure study, the level of knockdown was reduced even further, and very few bed bugs were knocked down in the double fabric study. The results of this study indicate that pyrethroid-impregnated mattress liners are not likely to be effective in the management of most modern bed bug infestations involving either C. hemipterus or C. lectularius.

Public Awareness, Perception, and Knowledge of Bed Bug Infestation Prevalence in Iraq.

Background: Bed bugs are blood-feeding insects and are an important urban pest. Bed bugs are nocturnal insects and hide in cracks in walls and beds during the day. The study aims to: (1) determine the bed bugs species that infest Iraq, their infestation source, and their distribution; (2) determine the level of awareness and concern regarding bed bugs among the Iraqi community. Methods: Between 2020 and 2021, a survey of bed bug infestation dynamics was conducted in 18 infested sites located in major cities across eight governorates. The 23-item online survey was distributed over social media to Iraqi citizens between June and July 2022 to survey on their familiarity with bed bug habitat, medical significance, transmission, prevention, control measures, and concerns. Results: There were a total of 1104 bed bugs collected. Based on the morphological characteristics, bed bugs collected from eight Iraqi governorates are identified as Cimex hemipterus. Besides, a high rate of bed bug infestation was observed in workers' dormitories. The online questionnaire was answered by 1843 respondents and based on the feedback, most respondents (55.3%) have no awareness of bed bug infestations, while around 43.8% are somewhat concerned about bed bug infestations in Iraq. Conclusion: Only tropical bed bugs, C. hemipterus were found in all sampled sites. Bed bug infestations are mainly caused by migrant workers and the reuse of second-hand furniture and clothing. The results suggest that the Iraqi government should organize more proper demonstrations on bed bug awareness for the public.

Testing the Sensitivity of the Tropical Bed Bug Cimex hemipterus (Hemiptera: Cimicidae) to Deltamethrin, Phoxim and Propetamphos in Eastern Iran.

Background: Bedbugs are nocturnal ectoparasites that have made a comeback after 20 years and have become one of the main challenges for pest control methods worldwide. Monitoring chemical pesticide resistance is crucial for identifying the best bed bugs management strategies to effectively manage arthropods. This study aims to assess the susceptibility of Cimex hemipterus (Hemiptera: Cimicidae) collected from different parts of Khorasan-Razavi Province, (northeast of Iran) to deltamethrin (pyrethroid), phoxim (organothiophosphate) and propetamphos (phosphoramidate). Methods: This study was conducted from Dec 2020 to May 2021. The efficacy of three insecticides (deltamethrin, phoxim, and propetamphos) on adult C.hemipterus was assessed using a bioassay method recommended by the World Health Organization (WHO),. Concentrations of deltamethrin used were 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 ppm, while concentrations of phoxim and propetamphos were 10, 40, 160, and 320 ppm. The bed bugs were continuously exposed to the insecticide for 24 hours, and mortality was assessed at regular intervals during the observation period. The concentration-response data were subjected to POLO-PC software and data were analyzed by the one-way and two-way ANOVA procedures. Results: The lethal concentration fifty values of deltamethrin, phoxim and propetamphos on the examined bed bugs were 0.551, 0.148 and 0.237 ppm, respectively. Insecticide effects of phoxim were significantly higher (P≤ 0.05) compared to each of either deltamethrin and propetamphos agents. Conclusion: The insecticide effects of phoxim against bed bugs were significantly higher compared to each of either deltamethrin or propetamphosinsecticides.

Metagenomic 16S rDNA amplicon data of microbial diversity of Cimex hemipterus (F.) (Hemiptera: Cimicidae) treated with insect growth regulators (IGR).

The metagenomics dataset presented here is based on bacterial 16S rDNA gene amplicons of DNA extracted from tropical bed bugs (Cimex hemipterus). Amplicon-based sequencing was performed using the Illumina MiSeq platform, and the raw sequence data were analyzed using QIIME (version 2022.8.3). The metagenome sequence comprised ten samples that include C1 (133 511bps), C2 (108 920bps), CH1 (106 562bps), CH2 (101 778bps), P1 (103 618bps), P2 (133 258bps), T1 (113 558bps), T2 (133 952bps), TM1 (125 335bps), and TM2 (118 345bps). The sequence data is readily accessible at the NCBI SRA under bio project PRJNA918835. The most abundant microbial community present in the C. hemipterus is the Proteobacteria, with more than 99% of the abundance.

Detection of Arthropod-Borne Bacteria and Assessment of MALDI-TOF MS for the Identification of Field-Collected Immature Bed Bugs from Mauritania.

Human infestations by bed bugs have upsurged globally in recent decades, including in African countries, where recent reports pointed out an increase in infestation. Sympatric dwelling has been described for two species of bed bug parasitizing humans: Cimex hemipterus (the tropical bed bug) and C. lectularius. Identification of these two species is based on morphological characteristics, and gene sequencing, and may also rely on Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS). The present work aimed to assess whether MALDI-TOF MS was applicable for species level identification of immature stages of Cimex. Arthropods were collected in domestic settings in Nouakchott, Mauritania. Identification used morphological keys and MALDI-TOF MS identification was assessed for immature stages. Quantitative PCR and sequencing assays were used to detect arthropod-associated bacteria in each specimen. A total of 92 arthropods were collected, all morphologically identified as C. hemipterus (32 males, 14 females and 45 immature stages). A total of 35/45 specimens produced good quality MALDI-TOF MS spectra. Analysis allowed species level identification of all immature C. hemipterus after their spectra were entered into our in-house MALDI-TOF MS arthropod spectra database. Molecular screening allowed detection of Wolbachia DNA in each specimen. These results suggested that MALDI-TOF MS is a reliable tool for species level identification of Cimex specimens, including immature specimens. Future studies should assess this approach on larger panels of immature specimens for different Cimex species and focus on the precise staging of their different immature developmental stages.

Analysis of Transcriptome Difference between Blood-Fed and Starved Tropical Bed Bug, Cimex hemipterus (F.) (Hemiptera: Cimicidae).

The reference transcriptome for Cimex hemipterus (tropical bed bug) was assembled de novo in this study, and differential expression analysis was conducted between blood-fed and starved tropical bed bug. A total of 24,609 transcripts were assembled, with around 79% of them being annotated against the Eukaryotic Orthologous Groups (KOG) database. The transcriptomic comparison revealed several differentially expressed genes between blood-fed and starved bed bugs, with 38 of them being identifiable. There were 20 and 18 genes significantly upregulated in blood-fed and starved bed bugs, respectively. Differentially expressed genes (DEGs) were revealed to be associated with regulation, metabolism, transport, motility, immune, and stress response; endocytosis; and signal transduction. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed an enrichment of genes encoding steroid biosynthesis, glycosaminoglycan biosynthesis, butanoate metabolism, and autophagy in both blood-fed and starved bed bugs. However, in blood-fed bed bugs, genes involved in histidine metabolism, caffeine metabolism, ubiquinone/terpenoid-quinone biosynthesis, and sulfur relay system were enriched. On the other hand, starvation activates genes related to nicotinate and nicotinamide metabolism, fatty acid elongation, terpenoid backbone biosynthesis, metabolism of xenobiotics by cytochrome P450, riboflavin metabolism, apoptosis, and protein export. The present study is the first to report a de novo transcriptomic analysis in C. hemipterus and demonstrated differential responses of bed bugs in facing blood-feeding and starvation.

Morphological, Molecular and MALDI-TOF MS Identification of Bedbugs and Associated Wolbachia Species in Rural Senegal.

Bed bugs are known to carry several microorganisms. The purpose of this study was to assess the prevalence of bed bug infestation in two rural areas of Senegal and determine the species present in the population. A screening was conducted to detect some arthropod associated pathogenic bacteria in bed bugs and to evaluate the prevalence of endosymbiont carriage. One survey took place in 17 villages in Niakhar and two surveys in Dielmo and Ndiop and surroundings area in the same 20 villages. Bed bugs collected were identified morphologically and by MALDI-TOF MS tools. Microorganisms screening was performed by qPCR and confirmed by sequencing. During the survey in the Niakhar region, only one household 1/255 (0.4%) in the village of Ngayokhem was found infested by bed bugs. In a monitoring survey of the surroundings of Dielmo and Ndiop area, high prevalence was found during the two rounds of surveys in 65/314 (21%) in 16/20 villages (January-March) and 93/351 (26%) in 19/20 villages (December). All bed bugs were morphologically identified as the species Cimex hemipterus, of which 285/1,637 (17%) were randomly selected for MALDI-TOF MS analysis and bacteria screening. Among the Bacteria tested only Wolbachia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) DNA was found in 248/276 (90%) of the bedbugs. We briefly describe a high level of non-generalized bed bug infestation in rural Senegal and the diversity of Wolbachia strains carried by C. hemipterus. This study opens perspectives for raising household awareness of bed bug infestations and possibilities for appropriate control.

Identification and Validation of Reference Genes for Expression Analysis Using qRT-PCR in Cimex hemipterus (Hemiptera: Cimicidae).

The tropical bed bug, Cimex hemipterus (F.) (Hemiptera: Cimicidae) is an important public-health pest that feeds on the blood of humans and some other animals. To explore the function of the target genes of C. hemipterus, it is essential to select suitable reference genes for the accurate quantification of gene expression. Here, we selected 10 frequently used reference genes in insects and evaluated their stability in C. hemipterus under various biotic (developmental stage, sex, and tissue) and abiotic (gas stimulation and temperature) conditions through RefFinder (which integrates four computational programs: geNorm, NormFinder, BestKeeper, and ∆Ct). Results indicate that the optimal combination of reference genes for each experimental condition was as follows: RPL8 and EF1α for the developmental stage (eggs, early instar nymphs, late instar nymphs, and adults), RPL8 and RPS16 for adult sex, RPL8 and RPL11 for adult tissue (head, thorax, abdomen, and legs), RPL8 and ß-tubulin for gas stimulation (air and carbon dioxide), and RPL8 and NADH for temperature (0, 5, 17, 30, and 37 °C). Finally, the expression pattern of the HSP70 and GR21 genes were analyzed, and the results highlight the importance of appropriate reference-gene selection. Our results provide a comprehensive list of optimal reference genes from C. hemipterus for the first time, which will contribute to accurately analyzing the expression of target genes.