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Journal of Medical Entomology, XX(X), 2020, 1–5
doi: 10.1093/jme/tjz247
Short Communication Short Communication
Cimex lectularius Linnaeus, 1758 (Hemiptera: Cimicidae) 
in Costa Rica: First Case Report Confirmed by Molecular 
Methods in Central America
Juan Carlos Cambronero-Heinrichs, 1,3 Laura Sofía Sánchez-Portilla, 2  
Ólger Calderón-Arguedas, 1 and Adriana Troyo1
1
AADate Centro de Investigación en Enfermedades Tropicales (CIET), Facultad de Microbiología, Universidad de Costa Rica, Sede Rodrigo 
Facio Brenes, Montes de Oca, San José, Costa Rica, 11501-2060, 2Escuela de Biología, Universidad de Costa Rica, Sede Rodrigo Facio 
Brenes, Montes de Oca, San José, Costa Rica, 11501-2060, and  3Corresponding author, e-mail: juan.cambroneroheinrichs@ucr.ac.cr
AAMonth
Subject Editor: Roberto Pereira
AAYear Received 11 September 2019; Editorial decision 2 December 2019
Abstract
Cimex lectularius and Cimex hemipterus are the most common species of bedbugs that infest homes. Although 
case reports decreased substantially by the end of the 20th century, bed bugs, and especially C. lectularius, 
are currently suffering a resurgence mostly attributed to insecticide resistance, inadequate pest control, and 
increased travel. Here, we report, to the best of our knowledge, the first molecular confirmation of C. lectularius 
in Central America. Specimens were obtained from an apartment located in Heredia, Costa Rica. These spe-
cimens were identified morphologically as C. lectularius. The species identification was confirmed by ampli-
fying and sequencing fragments of the cytochrome oxidase subunit I (COI) and the 16S rRNA (16S) genes. The 
phylogenetic analysis showed that the sequences obtained were more closely related to a C. lectularius mi-
tochondrial complete genome sequence from China, with similarities of 98.84% (686/694) for COI and 98.97% 
(387/391) for 16S. The finding of C. lectularius in Costa Rica will require further investigation in order to deter-
mine the extent of current infestations and the susceptibility to insecticides, especially due to the impact that 
this species can have in human health, as well as the tourism industry in the region.
Key words: Cimicidae, Cimex lectularius, bedbugs, Central America, Costa Rica
Cimicidae is a family of cosmopolitan insects that are temporary For example, both bedbugs are highly resistant to pyrethroids world-
but obligate hematophagous ectoparasites and feed at night from wide, and recent studies have revealed that pyrethroid-resistance–as-
hosts that mainly include humans, bats, and birds (Reinhardt and sociated mutations (kdr) are frequent in C. lectularius populations 
Siva-Jothy 2007, Zorrilla-Vaca et al. 2015) The species adapted to from Europe and the United States (Dang et  al. 2015, Balvin and 
feeding on human blood, known as bedbugs, are Cimex lectularius, Booth 2018, Holleman et al. 2019), and this evidence should influ-
which is found mainly in temperate latitudes, and Cimex hemipterus ence control strategies applied to new infestations.
Fabricius, 1803 (Hemiptera: Cimicidae) which is prevalent in tropical In the past decade, C.  lectularius infestations in the Americas 
and subtropical regions (Koganemaru and Miller 2013). This para- were registered in many countries, including Canada, United States, 
sitic relationship has been registered even in millennia-old Egyptian Venezuela, Peru, Argentina, and Chile (Zorrilla-Vaca et  al. 2015). 
archeological sites (Panagiotakopulu and Buckland et al. 1999), be- During that same period of time, there were no published records 
coming widespread and common all over the world in the first half of bed bug occurrence in Central America, with the exception of a 
of the 20th century (Potter 2011). With the discovery and repeated single report from Nicaragua, where no species identification was 
use of insecticides such as DDT (dichloro-diphenyl trichloroethane), conducted (Bendaña-García 2018). Here, we present the first report 
bed bug infestations declined dramatically by the end of the 20th of a C.  lectularius infestation confirmed by molecular methods in 
century (Doggett et al. 2012). Unfortunately, bedbugs, and especially a property in Costa Rica and Central America, which represents 
C. lectularius, have resurged notably over the past decade in many a case of bed bug resurgence in an area where C. hemipterus was 
areas of the world, mostly due to an increase in insecticide resistance, the prevalent species. This finding may represent the initial stages 
inadequate pest control, and international travel (Dang et al. 2017). of invasion, although it is also possible that other infestations are 
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2 Journal of Medical Entomology, 2020, Vol. XX, No. XX
being misidentified as C. hemipterus, given its predominantly trop- DNA Extraction and Molecular Analyses
ical distribution. One nymph was macerated in a microcentrifuge tube using a sterile 
pestle and resuspended in 1 ml of sterile saline solution (0.9 % NaCl). 
Methods Genomic DNA extraction was carried out using the NucleoSpin 
Tissue Kit (Macherey-Nagel), according to the manufacturer’s in-
Insect Sampling and Morphological Identification structions. For molecular identification, one-step PCR protocols were 
Bedbugs were obtained from crevices of a bed at an apartment in employed to amplify fragments of two mitochondrial genes: the cy-
Heredia, Costa Rica, in June 2017. There, the inhabitants reported tochrome oxidase subunit I (COI) gene, using primers LepF (5′-ATT 
a rash in feet and hands and insomnia, as a result of the insect bites. CAA CCA ATC ATA AAG ATA TNG G-3′) and LepR (5′-TAW ACT 
Live insects were flushed out by heating the piece of wooden furni- TCW GGR TGT CCR AAR AAT CA-3′; 712 bp product); and the 
ture with a hairdryer. Specimens that came out were collected and 16S rRNA gene (16S), using primers LR-J-13007 (5′-TTA CGC TGT 
stored in 70% ethanol at −20°C, prior to identification. Insects were TAT CCC TAA-3′) and LR-N-13398 (5′-CGC CTG TTT ATC AAA 
identified morphologically by direct observation of key characters AAC AT-3′; 391 bp product; Balvín et al. 2012). PCR products were 
(Fig. 1) under a dissecting stereo microscope (CDC 1967, Campbell purified using ExoSAP-IT (Applied Biosystems), and they were sent to 
et al. 2016). Macrogen Inc. (Seoul, South Korea) for sequencing. DNA sequences 
Fig. 1. Cimex lectularius specimens collected in Heredia, Costa Rica. (a) Dorsal view of a female adult, (b) head and pronotum with a deeply excavated anterior 
margin, (c) ventral view of female abdomen showing the paragenital sinus, (d) ventral of a male adult, and (e) dorsal view of a nymph.
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Journal of Medical Entomology, 2020, Vol. XX, No. XX 3
were edited and assembled with DNA Baser (Heracle BioSoft), and of the COI and 16S genes from one specimen. The partial sequences 
they were identified by sequence homology to publically available of COI (712 bp) and 16S (391 bp) obtained showed high similarity 
sequences using the Basic Local Alignment Search Tool (BLAST). on BLAST to C. lectularius sequences from Europe and China (Table 
Sequences obtained were deposited in GenBank with Accession num- 1). Moreover, the phylogenetic analysis with concatenated sequences 
bers MN265385 (16S) and MN271345 (COI). indicated that specimens collected in Costa Rica were more closely 
A concatenated phylogenetic tree of both 16S and COI genes related to C.  lectularius from China (Fig. 2). However, it is impor-
(Fig. 2) and two individual trees (Supp Material [online only]) for tant to note that among the sequences of C. lectularius in GenBank, 
both genes were constructed as described by Cambronero-Heinrichs none of them are from countries of Latin America. Furthermore, we 
et al. (2019). Briefly, existing Cimex spp. sequences were retrieved want to highlight that to avoid mis-identification morphological and 
from GenBank, sequence alignment was done separately for every barcoding identification should be conducted together.
gene on the MAFFT 7 CRBC Server, the aligned sequences were ad- Results show that C.  lectularius is present in Costa Rica. 
justed to the same length in Mega 7, and they were concatenated Although this species has a cosmopolitan distribution, it has been 
manually. The phylogenetic analysis was performed employing the found mainly in temperate regions of the world (Zorrilla-Vaca et al. 
Bayesian method with Mr. Bayes software, using 2 million genera- 2015), whereas the tropical bed bug, C.  hemipterus, was the one 
tions and setting the burn-in fraction to 0.25. The Bayesian informa- previously reported throughout tropical and subtropical regions, in-
tion criterion was selected, and final editions of the tree were made cluding Central America (Usinger 1966). Moreover, this infestation 
using Mega7. outside of the assumed range of this species is comparable to reports 
of C. hemipterus on continental North America, outside what is con-
sidered its natural range (Campbell et al. 2016).
Historical evidence from Central America confirms that 
Results and Discussion
C.  hemipterus was the species prevalent in the region, including 
In total, 25 bed bug specimens, 16 nymphs and 9 adults (4 males and 5 Nicaragua, Costa Rica, and Panama (Usinger 1966). Moreover, pre-
females), were collected and identified preliminarily as C. lectularius, vious to this report, the Collection of Medical Entomology at the 
based on the presence of a pronotum with a deeply excavated anterior University of Costa Rica had only C. hemipterus specimens from dif-
margin (Fig. 1; CDC 1967, Campbell et al. 2016). This morpholog- ferent areas of the country, and most were obtained before the 1980s 
ical identification was confirmed by sequencing of partial fragments (the most recent specimen was collected in 1998; Adriana Troyo, 
Fig. 2. Phylogenetic analysis based on the concatenated COI and 16S gene fragments of selected Cimex spp. sequences (GenBank) and C. lectularius from Costa 
Rica (this study). Scale bars represent number of substitutions per nucleotide position. The numbers above the branches represent their Bayesian-calculated 
posterior probabilities. For individual trees for both genes see Supp Figs. S1 and S2 (online only).
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4 Journal of Medical Entomology, 2020, Vol. XX, No. XX
Table 1. Similarity of Cimex lectularius from Costa Rica (CR2017) with close relative sequences from GenBank
ID Country 16S  COI  
% similarity (bp) % similarity (bp)
Complete Genome China 98.97 (387/391) 98.84 (686/694)
26 Czech Republic 99.47 (377/379) 99.09 (652/658)
39 Czech Republic 99.47 (377/379) 99.09 (652/658)
110 France 99.21 (376/379) 99.24 (653/658)
133 Serbia 99.47 (378/380) 98.78 (649/657)
750 Italy 99.21 (377/380) 98.18 (646/658)
personal communication). There have been no confirmed reports of for all the help during sampling. We thank Brittany Campbell for providing 
bed bug infestations in Costa Rica for more than 20 yr, until now. helpful information and guidance for morphological identification of species. 
The global resurgence of Cimex spp. was reviewed by Zorrilla- This research was funded in part by Universidad de Costa Rica projects B5735 
Vaca et al. (2015), and they show that recent reports from all over and ED548. The authors declare no conflict of interest.
the world follow a similar pattern as to the historical distribution of 
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