First record of Ornithocoris pallidus (Hemiptera: Cimicidae) associated with Hyacinth macaw (Anodorhynchus hyacinthinus) nest in the Brazilian Pantanal

Abstract

This study reports the first record of Ornithocoris pallidus associated with a Hyacinth Macaw (Anodorhynchus hyacinthinus) nest in the Brazilian Pantanal supported by integrative morphological and molecular identification. Ectoparasites associated with nests can compromise the health and development of birds that use reused cavities, such as the hyacinth macaw. Among these parasites, blood-feeding bugs of the Cimicidae family remain poorly studied in wild environments. Accordingly, the specimens were collected in February 2025 from a natural cavity containing a blue macaw chick and from a nest box occupied by muscovy duck (Cairina moschata). Identification was performed using light microscopy and scanning electron microscopy, following the diagnostic taxonomic key, as well as partial sequencing of the cytochrome c oxidase subunit I (COI) gene. The observed morphological characteristics were consistent with Ornithocoris pallidus, including the presence of 1 + 1 long setae on the posterolateral margin of the pronotum, which is a diagnostic taxonomic character for the species. The obtained sequence (306 bp) showed 96.73% identity with sequences available in GenBank for the species. The results expand our knowledge of the distribution and host range of O. pallidus, in addition to contributing to previously unpublished molecular data on cimicids associated with Neotropical avifauna, reinforcing the importance of integrating the monitoring of threatened birds with parasitological assessment.

Graphical abstract

Highlights

• •First record of Ornithocoris pallidus developing in the nesting cavities of Anodorhynchus hyacinthinus.
• •Integrative identification using stereomicroscopy, scanning electron microscopy (SEM), and molecular methods.
• •Expands morphometric and molecular data on Cimicidae in wild avifauna.

1. Introduction

Ectoparasites are organisms frequently associated with wild birds and can influence important aspects of their hosts’ biology, such as health, development, and reproductive success (Janiszewska et al., 2025; Gutiérrez López et al., 2026). Species associated with nests deserve special attention, since these environments function as stable microhabitats, favoring the persistence, reproduction, and transmission of parasites, especially during the breeding season (Hansell, 1993; Merino et al., 2025; Mazorra-Alonso et al., 2024).

Among avian ectoparasites, insects of the Cimicidae family are particularly noteworthy; these hematophagous hemipterans predominantly infest birds but can occasionally parasitize mammalian hosts these are blood-feeding bed bugs that can also parasitize mammals (Usinger and Matsuda, 1959; Akhoundi et al., 2020; Bastos et al., 2021; Hamlili et al., 2023). Characterized by cryptic habits, these insects harbor within crevices, structural cavities, and nesting substrates, foraging primarily during nocturnal periods (Forattini, 1990). Although widely known in urban contexts, cimicids associated with wildlife remain understudied (Usinger and Matsuda, 1959; Forattini, 1990; Bartonička and Gaisler, 2007).

The Ornithocoris genus includes species of cimicids predominantly associated with wild birds (Usinger and Matsuda, 1959; Di Iorio et al., 2010). Ornithocoris pallidus was originally described in association with swallow nests, which led to its recurring association with this group of birds (Usinger and Matsuda, 1959; Di Iorio et al., 2010). However, evidence indicates that the species is not host-specific and may occur on different bird species, particularly those that use cavities or reused nests (Reinhardt and Siva-;Jothy, 2007). Nevertheless, information on its biology, distribution, and genetic variability remains limited.

Molecular tools have proven to be important for taxonomic and phylogenetic studies, as well as for the identification of cryptic species (Hebert et al., 2003). The cytochrome c oxidase 1 (COI) gene, used in DNA barcoding, is an important marker for species delineation, especially in groups with conserved morphology (Hebert et al., 2003; Balvín et al., 2015). However, despite its well-established use across various taxa, the application of this marker within the Cimicidae family remains limited, particularly for species associated with wild birds and wild populations in isolated environments such as the Pantanal (Reinhardt and Siva-Jothy, 2007; Balvín et al., 2015).

The hyacinth macaw (Anodorhynchus hyacinthinus) is an emblematic species of Brazilian fauna, threatened with extinction, of great ecological and conservation significance, that nests in cavities that may be used by different species over consecutive years (Guedes and Harper, 1995; Fontoura, 2023; BirdLife International, 2025). This prolonged occupation promotes the formation of stable microenvironments capable of sustaining communities of ectoparasites and facilitating their persistence and potential transmission among hosts (Heeb et al., 2000; Carvalho et al., 2005; Cockle et al., 2010; Fontoura, 2023). In seasonally flood-prone environments, such as the Pantanal, high humidity and elevated temperatures can intensify these processes, contributing to the diversity and abundance of these organisms (Marshall, 1981; Bartonička and Gaisler, 2007).

Despite previous reports of O. pallidus associated with birds, no integrative morphological and molecular characterization has been reported for specimens collected from hyacinth macaw nests in the Brazilian Pantanal. Furthermore, molecular data for Neotropical cimicids remain scarce in public databases, limiting the precise delineation of species and assessments of intraspecific variability. Therefore, this study aimed to provide the first record of O. pallidus collected from hyacinth macaw nests in the Pantanal, integrating morphological, morphometric, and molecular characteristics, thereby contributing to the understanding of the geographic distribution of Cimicidae associated with wild birds in isolated environments.

2. Materials and methods

2.1. Study area and sampling

Ectoparasite samples were collected in the Miranda subregion of the Pantanal, Mato Grosso do Sul, Brazil (19°57′56″S, 56°18′37″W), during nest monitoring conducted by the Arara Azul Project (Arara Azul Institute, Brazil) (Fig. 1). The Pantanal is a vast seasonal floodplain characterized by annual flood and dry cycles that shape its ecological dynamics. The Miranda subregion features a mosaic of vegetation types and high biodiversity.

Fig. 1.

Cimicid specimens preparation workflow for scanning electron microscopy (SEM) examination.

The recurring presence of blood-feeding ectoparasites in nests had been previously recorded since 2014, but without taxonomic identification. In February 2025, 16 specimens were collected from two separate nests: (i) a natural cavity containing a developing hyacinth macaw (Anodorhynchus hyacinthinus) chick and (ii) a nest box occupied by a muscovy duck (Cairina moschata). In the nest with the blue macaw chick, the chick was handled for biometric procedures, during which an external physical examination was performed to detect ectoparasites. The chick's age was determined based on the known hatching date, since the nest was monitored during the breeding season. The egg hatched on October 17, 2024, and the chick was subsequently monitored longitudinally until it fledged. The interior of the nests was also inspected for the presence of hematophagous arthropods.

Cimicid collection was conducted on an opportunistic basis during the period set aside for nest monitoring and management, with the aim of obtaining specimens for taxonomic identification. Sampling was not designed to estimate the abundance of ectoparasites or the intensity of infestation. Consequently, not all visible specimens were collected, and the time and effort spent searching were not standardized across nests. Collection was carried out with a focus on expediting management and minimizing the time spent on nest interventions.

The specimens were removed manually and individually placed in 1.5 mL microtubes. A total of 16 specimens were collected, eight from the hyacinth macaw nest and eight from the muscovy duck nest. The 16 collected specimens were distributed equally between the two nest types (n = 8 per source) and processed according to the analysis technique as shown in Table 1.

Table 1.

Detailed information on the storage of specimens, including the quantity collected from each nest, how they were preserved, analyzed and stored.

N	Nests	Preservation	Screening test	SEM	DNA extraction	PCRa	Storage
1	Natural cavity	10% formalin	Stereomicroscope	No	No	No	ZUFMS-HEMO1844
2	Natural cavity	70°GL ethyl alcohol	KOH	No	No	No	Consumed
3	Natural cavity	70°GL ethyl alcohol	Stereomicroscope	Yes	No	No	Consumed
4	Natural cavity	70°GL ethyl alcohol	Stereomicroscope	No	Yes	Yes	Consumed
5	Natural cavity	70°GL ethyl alcohol	Stereomicroscope	No	No	No	Morphometry
6	Natural cavity	70°GL ethyl alcohol	Stereomicroscope	No	No	No	Morphometry
7	Natural cavity	70°GL ethyl alcohol	Stereomicroscope	No	No	No	Morphometry
8	Natural cavity	70°GL ethyl alcohol	Stereomicroscope	No	No	No	Morphometry
9	Nest box	10% formalin	Stereomicroscope	No	No	No	ZUFMS-HEMO1844
10	Nest box	70°GL ethyl alcohol	KOH	No	No	No	Consumed
11	Nest box	70°GL ethyl alcohol	Stereomicroscope	Yes	No	No	Consumed
12	Nest box	70°GL ethyl alcohol	Stereomicroscope	No	Yes	Yes	Consumed
13	Nest box	70°GL ethyl alcohol	Stereomicroscope	No	No	No	Morphometry
14	Nest box	70°GL ethyl alcohol	Stereomicroscope	No	No	No	Morphometry
15	Nest box	70°GL ethyl alcohol	Stereomicroscope	No	No	No	Morphometry
16	Nest box	70°GL ethyl alcohol	Stereomicroscope	No	No	No	Morphometry

^asamples that were sent for PCR were subjected to capillary electrophoresis sequencing.

The final taxonomic identification was performed by integrating molecular and imaging data, using the dichotomous key proposed by Usinger (1966). The specimens were collected under authorization N°. 83255-6 (SISBIO).

2.2. Morphological identification

The morphometric parameters were analyzed using a Leica M205 A™ stereomicroscope equipped with a Leica DFC 420™ camera and documented and measured using the LEICA Application Suite - LAS™ 3.8 image analysis system (Leica Microsystems™, Wetzlar and Mannheim, Germany). The parameters analyzed were body conformation, coloration, total length, head length, head width, head width including the eyes, interocular distance, pronotum height, pronotal bristle, and the lengths of the 2nd, 3rd, and 4th antennal segments. This stage allowed a preliminary morphological screening and the observation of diagnostic taxonomic characters present in the specimens. However, it is important to note that the condition and limited number of specimens imposed significant restrictions on the morphometric analyses. Many individuals were degraded, with the loss of key setae and altered diagnostic parameters, making it impossible to fully measure all structures.

Given that the diagnostic taxonomic characteristics of the species are subtle, particularly those related to the morphology of the pronotum and the distribution pattern of the lateral setae, it was necessary to use scanning electron microscopy (SEM) for a more detailed analysis.

Subsequently, for the preparation of the SEM, one specimen from each nest preserved in 70° GL ethanol was prepared at the Laboratório Multiusuário de Captura de Imagens do Instituto de Biociências at the Federal University of Mato Grosso do Sul (INBIO - UFMS). The specimens were subjected to a series of ethyl alcohol solutions in increasing concentrations (70, 80, 90° GL, and absolute), with each concentration maintained for 24 hours (Gordh and Hall, 1979). After the series of increasing alcohol concentrations, the specimen was transferred to a solution of absolute alcohol and hexamethyldisilazane (HDMS) (1:1 v/v) for 10 minutes. The specimen was then placed on a carbon-conductive adhesive tape (PELCO Tabs™, Carbon Conductive Tabs, 12 mm OD; Ted Pella, Inc., USA), attached to a metal stub. After drying, the material was examined using a TM 3000 scanning electron microscope (Hitachi HighTech Corporation, Japan), and images were acquired in Analy mode at magnifications ranging from ×40 to ×300, depending on the morphological region under examination (Fig. 3). The assay workflow is illustrated in Fig. 1.

Fig. 3.

Stereomicroscope and scanning electron microscope (SEM) images of Ornithocoris pallidus. (A) Ventral view of the specimen, highlighting the thoracic structures and the arrangement of the antenna segments, thoracic structures, eye arrangement, and robust femur of the third pair of legs. (B) Ventral view of the head, highlighting the thoracic structures and the arrangement of the proboscis. (C) Detail of the posterolateral margin of the pronotum, showing the long lateral setae 1 + 1, whose projections extend beyond the posterior margin of the pronotum (arrow). (D) Higher-magnification detail of the posterolateral region of the pronotum, highlighting the insertion, orientation, and morphology of the long lateral setae 1 + 1 of the pronotum (arrows). (E) Dorsal view of the head and pronotum (pro) region showing the laterally projecting eyes and the lateral setae extending beyond the posterior margin of the pronotum. (F) Ventral view highlighting the long lateral setae of the pronotum 1 + 1, a 4-segmented antenna, and eyes with ommatidia and interfacetal setae (arrow).

Taxonomic identification was conducted using the dichotomous keys proposed by Forattini (1990) for subfamily Haematosiphoninae and genus Ornithocoris, and Usinger (1966) for species level.

2.3. Molecular identification

DNA was extracted from two specimens (one originating from the Anodorhynchus hyacinthinus nest and one from the Cairina moschata nest), and subsequently subjected to polymerase chain reaction (PCR) amplification, which were conducted at the Molecular Biology Laboratory, School of Veterinary Medicine and Animal Science (FAMEZ-UFMS). Extraction was performed using an adapted, automated method with the Maxwell® RSC instrument (Promega) in conjunction with the Maxwell® RSC PureFood GMO and Authentication Kit (Catalog No. AS1600; Promega Corporation, Madison, WI, USA).

In summary, the specimens were individually placed in microtubes containing 500 μL of phosphate-buffered saline (PBS) and 10 μL of proteinase K. The tubes were then vortexed for 30 seconds for initial homogenization. After this step, the samples were incubated at 65°C for 10 minutes, followed by cooling to room temperature for 2 minutes. In order to enhance the lysis process, glass beads were added to each sample, which were then vortexed again for 1 minute.

The preparation of the cartridges and the execution of the protocol on the Maxwell® RSC instrument (Promega Corporation, USA) were performed according to the manufacturer's instructions. At the end of the procedure, the DNA was eluted into 50 μL of elution buffer (ultrapure water) and stored at −20°C until used in subsequent analyses.

The cytochrome c oxidase subunit I (COI) gene was selected as the molecular target. PCR reactions were performed in a final volume of 20 μL containing 10 μL of GoTaq Green Master Mix 2× (Promega, USA), 10 μM of each primer, approximately 50 ng of DNA, and ultrapure water to 20 μL.

The primers Lep1F (5′ - ATT CAA CCA ATC ATA AAG ATA TTG G - 3′) and Lep3R (5′ - TAT ACT TCA GGG TGT CCG AAA AAT CA - 3′) were used, as described by Balvín et al. (2015); these primers amplify a 658-base pair (bp) fragment widely used in the molecular identification of different insect orders. DNA from Rhipicephalus sanguineus obtained was used as positive control (Soares et al., 2020). Sterilized ultrapure water was used as negative control. Amplification conditions consisted of an initial denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation at 94°C for 40 s, annealing at 53°C for 1 min, and extension at 72°C for 1 min, with a final extension step at 72°C for 10 min.

Amplified products were subjected to electrophoresis in 2% agarose gel stained with GelRed® Nucleic Acid Gel Stain (Biotium, San Francisco, CA, USA) at 90 V/150 mA for 45 min. Bands were visualized under ultraviolet light using the Easy Doc 100 documentation system (Bio Agency, São Paulo, Brazil).

The PCR product was purified using ExoSAP-IT™ Express PCR Product Cleanup reagent (Applied Biosystems™). Sequencing was then performed using the chain-termination method (Sanger et al., 1977) with the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific™, Waltham, MA, USA), and sequences were read using a SeqStudio automated DNA analyzer (Applied Biosystems™) at the Laboratório de Virologia Veterinária, FAMEZ-UFMS.

Generated sequences were quality-checked in Geneious Prime version 2026.0.2 (Kearse et al., 2012) by manual inspection of electropherograms, removal of low-quality regions, and construction of a consensus sequence.

3. Results

3.1. Specimen collection

In the natural cavity, cimicids were observed both attached to the skin of the hyacinth macaw chick and scattered throughout the natural cavity and at its entrance (Fig. 2). In the nest box, there were four dead chicks of muscovy duck and two unviable eggs at the time of sampling. Cimicids were collected from the nest box and were observed on the dead chicks and the eggs. No direct feeding behavior was observed.

Fig. 2.

Cimicids attached to the skin of a hyacinth macaw (A. hyacinthinus; estimated age, 103 days, photographed in January 2025) nestling (A) and several specimens dispersed at the entrance of a natural nest cavity (B, collected in February 2025) in the Miranda Pantanal, Mato Grosso do Sul, Brazil.

The hyacinth macaw chick was monitored longitudinally from hatching on October 17, 2024, until it left the nest at 133 days of age. During the monitoring period, its physical and plumage development appeared to be slower compared to other chicks of similar age monitored by the team. During the same period, a high presence of cimicids was observed both on the chick and in the nest material. Despite this, the chick completed its development, made its first flight, and was subsequently observed after leaving the nest.

3.2. General morphology and taxonomic identification

Cimicid specimens collected from nests were identified as Ornithocoris pallidus based on external morphological characters observed under light microscopy and scanning electron microscopy (SEM), in accordance with the classical taxonomic criteria. Identification followed the dichotomous key proposed for the Hematosiphoninae subfamily, initially considering diagnostic characters at the generic level, as widely adopted in taxonomic studies of bird-associated cimicids (Usinger, 1966; Bastos, 2016).

The morphometric analysis was performed on a subset of viable specimens collected from both the natural cavity and the artificial nest box, although the quality of preservation limited the measurement of all parameters in all individuals. Adult females (n = 3) had a mean total length of 2.988 mm (range 2.255–4.435 mm), notably specimen C1 (4.435 mm), whose dimensions exceed laboratory measurements and resemble records of wild populations in the literature (Usinger, 1966; Bastos, 2016). For this group, the width of the head including the eyes averaged 0.779 mm, and the average interocular distance was 0.530 mm. Regarding the males (n = 3), the average length was 2.044 mm (range from 2.033 to 2.067 mm). Diagnostic parameters such as the mean interocular distance were 0.389 mm, and the mean length of the 4th antennal segment was 0.261 mm in the least degraded specimens.

These specimens (evaluated n = 6 adults) are also notable for the presence of dense, long setae on the protonum (1 + 1), a key diagnostic characteristic used in the dichotomous key. The pronotum has gently rounded lateral margins, with a long bristle inserted in the posterolateral region (1 + 1) measuring 0.128-0.197 mm, whose projection clearly extend beyond the posterior margin of the pronotum, as observed in the dorsal view and highlighted in the stereomicroscope and SEM images (Fig. 3). Crucially, the pronotum-to-head width ratio in our specimens reneged from 1.60 to 1.70 (mean 1.65), remaining consistently below the 1.80 threshold. These diagnostic features, along with the specific taxonomic keys, couplets used, and a detailed differential diagnosis from Ornithocoris toledoi, are summarized in Table 2.

Table 2.

Morphological and morphometric comparison of the Ornithocoris specimens collected in this study with reference data for Ornithocoris pallidus and Ornithocoris toledo.

Diagnostic Characters	Nest specimens collected	Ornithocoris pallidus	Ornithocoris toledoi
Pronotum-to-Head Width Ratio	1.65 (range: 1.60 - 1.70)	1.60 - 1.70 (Usinger, 1966)	> 1.80 (Forattini, 1990)
Posterolateral Protonal setae	1 + 1 (confirmed by SEM)	1 + 1 (Usinger, 1966)	2 + 2 (Forattini, 1990)
Total length of females	4.435 mm (stereomicroscope)	≈4.00 mm (Usinger, 1966)	3.94 - 4.20 mm (Forattini, 1990)
Spermalege (female)	Dorsal, right	Dorsal, right	Dorsal, right

The head region is short and broad, with well-developed, laterally protruding compound eyes. The antennae consist of four segments, attached laterally in front of the edge of the eyes, with sensory bristles distributed along the antennae segments. The rostrum is short, robust, and directed ventrally, characteristic of blood-feeding cimicids (Usinger, 1966).

In the ventral view, the body is oval and dorsoventrally flattened, with a broadly expanded abdomen exhibiting well-defined abdominal segments covered by uniformly distributed microtrichia. The lateral margins of the abdomen are slightly convex, without evident spiny projections, consistent with previous descriptions of the species. The legs have robust femora and tibiae, with scattered bristles. Specimens not processed during the diagnostic stages were deposited in the Zoological Collection of the Federal University of Mato Grosso do Sul (ZUFMS) under accession number ZUFMS-HEM01844.

3.3. Molecular analysis

The cytochrome c oxidase subunit I (COI) gene fragment was successfully amplified and sequenced from the specimen originating from the Anodorrhynchus hyacinthinus nest. The resulting product was 306 bp long. PCR amplification did not yield successful results for the specimen originating from the Cairina moschata nest.

Comparison of the consensus sequence against the GenBank database using BLASTn revealed 96.73% nucleotide identity with previously deposited O. pallidus sequences (accession numbers MG596827.1 and MG596828.1). The sequence generated in this study was deposited in GenBank under accession number PZ033109.

4. Discussion

This study provides the first record of Ornithocoris pallidus associated with hyacinth macaw (A. hyacinthinus) nest in the Brazilian Pantanal, expanding the known host range and ecological context of this species of the Cimicidae family in the wild. Morphological identification confirmed diagnostic characters consistent with classical descriptions, particularly the presence of long setae on the posterolateral margin of the pronotum, as well as the shape of the head, antennae, and rostrum, which are compatible with Ornithocoris (Usinger, 1966; Bastos, 2016).

The use of scanning electron microscopy was crucial for documenting relevant microstructures in a group characterized by well-preserved morphology, thereby reducing the diagnostic ambiguities frequently reported for Cimicidae associated with wild birds (Reinhardt and Siva-Jothy, 2007; Balvín et al., 2015). Despite the limitations imposed by the small number of specimens and their state of preservation, rigorous application of the dichotomous key was essential for species identification. Confirmation was based on decisive characters, notably the presence of a pair of long setae (1 + 1) on the posterolateral margin of the pronotum (Usinger, 1966; Forattini, 1990). Additionally, the measured morphometric parameters, such as total length exceeding that of laboratory-reared colonies (Bastos, 2016; Bastos et al., 2021), are similar to the descriptions of wild populations reported by Usinger (1966), reflecting the reality of specimens collected in isolated environments in the Brazilian Pantanal.

Molecular characterization based on the mitochondrial COI gene corroborated the morphological identification. Although the identity value (∼96.73%) is below the threshold commonly used in DNA barcoding studies, this result should be interpreted in light of the low representation of wild cimicid sequences in public databases, especially for the genus Ornithocoris, which primarily comes from wild populations in isolated environments of the Pantanal, including the hyacinth macaw nests.

In taxonomically undersampled groups, values below 98% may reflect gaps in reference data, regional intraspecific variation, or population structure that has not yet been documented (Hebert et al., 2003; Foottit et al., 2008). The consistency between the diagnostic morphological characters and the best alignment obtained via BLASTn supports the assignment to O. pallidus, while also highlighting the need to expand the molecular database for Cimicidae.

The presence of cimicids both on the live hyacinth macaw chick and inside the natural cavity demonstrates that these ectoparasites can be present both in direct contact with the host and in the nest environment. In the nest box occupied by muscovy duck (C. moschata), cimicids were found inside the cavity, including on dead chicks and nonviable eggs. Although this observation may indicate an association between the cimicids and the biological material present in the nest, no feeding behavior or hemophagy was directly observed. Therefore, the presence of cimicids in this nest box does not confirm muscovy duck as a host; this occurrence should be interpreted as an association with the nest environment and the biological material available within the cavity.

This pattern emphasizes the importance of the availability and reuse of nesting sites as key factors in the maintenance of nesting populations of cimicids. Cavities used for several years, as in the case of hyacinth macaw, constitute relatively stable microenvironments, favoring the interannual persistence of hematophagous ectoparasites (Heeb et al., 2000; Cockle et al., 2010). In the Pantanal, high humidity and high seasonal temperatures may further enhance the development and survival of these insects (Marshall, 1981; Bartonička and Gaisler, 2007), contributing to their persistence in seasonally flood-prone environments.

Although the severity of the infestation was not determined, the presence of cimicids in active nests underscores the importance of incorporating the parasitic component into long-term monitoring programs for threatened birds. Blood-feeding ectoparasites can harm chicks through blood loss, increased physiological stress, and the potential transmission of infectious agents (Clayton et al., 2010; Martínez-de la Puente et al., 2011; Dudek et al., 2021).

Despite the presence of cimicids in the nest, the hyacinth macaw chick completed its development and made its first flight, and was subsequently observed after leaving the nest. However, its 133-day stay in the nest was longer than the average of approximately 104–107 days reported for normal-sized blue macaw chicks in the Brazilian Pantanal (Guedes et al., 2022), and exceeded the longest stay reported for individuals classified as “dwarfs” in that study (126 days).

Hence, the field team observed slower body and plumage development compared to other chicks of similar age, along with a high prevalence of cimicids on the chick and in the nest material. Considering the monitoring since hatching, the high presence of these ectoparasites, and the observed developmental pattern, the cimicid load may have contributed to the slower development and the prolonged period spent in the nest. Although this interpretation is biologically plausible, no standardized quantitative assessments of parasite load or development were conducted; therefore, the possible relationship between these factors should be investigated in future studies.

During the monitoring, researchers suffered frequent bites from O. pallidus, which resulted in persistent local inflammation. Given the confirmed identification of Ornithocoris pallidus and the absence of other hematophagous arthropods in the cavities, the lesions are consistent with opportunistic blood feeding by this species.

The sampling of O. pallidus was conducted in a qualitative and opportunistic manner during routine monitoring by the Hyacinth Macaw Project, strictly prioritizing nestling welfare and rapid field handling, typically limited to 20 minutes, to minimize human interference (Guedes et al., 2022). It is noteworthy that, despite the identification of this ectoparasite, no sanitary interventions or chemical treatments have been performed in natural cavities or nest boxes. This policy aims to preserve the biological integrity and natural behavior of the diverse species complex utilizing these cavities, which function as stable microhabitats. Maintaining the environment in its natural state allows for fundamental ecological processes to persist, such as the natural predation of these bugs by other invertebrates cohabiting the nest material, ensuring the balance of the "nest-cavity web" in the Pantanal.

Nest-dwelling cimicids are capable of exploiting alternative hosts when exposed to new blood sources, including humans in occupational settings (Usinger, 1966; Reinhardt and Siva-Jothy, 2007; Balvín et al., 2015). Persistent skin reactions following bed bug bites are also widely described in the literature, reflecting an inflammatory response to blood feeding (Reinhardt and Siva-Jothy, 2007; Doggett et al., 2018). This finding expands our ecological understanding of O. pallidus and highlights the interface between wildlife and human health, reinforcing the relevance of this topic from a One Health perspective.

5. Conclusion

This study presents the first record of Ornithocoris pallidus in hyacinth macaw (Anodorhynchus hyacinthinus) nests in the Pantanal, supported by integrative morphological and molecular identification. The application of integrative taxonomy methods, combining classical and molecular methods, provides a reliable basis for species identification and helps fill gaps in the molecular characterization of Cimicidae associated with avifauna. The results expand our understanding of the geographic distribution, host range, and importance of reused nests as microhabitats for blood-feeding ectoparasites, providing a foundation for future phylogenetic, ecological, and epidemiological studies.

CRediT authorship contribution statement

Ivanise Paula Sobota: Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Investigation, Conceptualization. Maria Eduarda Monteiro Nascimento: Writing – original draft, Methodology, Investigation, Conceptualization. Fernanda Fontoura: Methodology, Formal analysis, Conceptualization. Ana Cecília de Paula Lourenço Rocha: Resources, Methodology, Investigation. Vitor Morais de Oliveira: Writing – original draft, Visualization, Investigation. Herbert Patric Kellermann Cleveland: Writing – review & editing, Validation, Supervision, Methodology. Leila Sabrina Ullmann: Writing – review & editing, Validation, Supervision. Carlos Alberto Nascimento Ramos: Writing – review & editing, Writing – original draft, Supervision, Formal analysis, Data curation. Rafael Antonio do Nascimento Ramos: Writing – review & editing, Writing – original draft, Supervision, Data curation. Fernando Paiva: Writing – review & editing, Writing – original draft, Visualization, Supervision, Methodology, Investigation, Formal analysis. Neiva Maria Robaldo Guedes: Writing – review & editing, Writing – original draft, Validation, Supervision, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Data availability

The COI sequence generated in this study is available in GenBank under accession number PZ033109. Additional data are available from the corresponding author upon reasonable request, subject to institutional and permit restrictions.

Declaration of generative AI and AI-assisted technologies in the manuscript preparation process

During the preparation of this work, the authors used OpenAI ChatGPT to support translation from Portuguese into English and to improve language, readability, and journal formatting. The authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

Funding

Nest data collection was supported by Instituto Arara Azul through sponsorship from Fundação Toyota do Brasil, Zurich Zoo, and the Adote um Ninho campaign. The role of the sponsors in study design, data collection, analysis, interpretation, manuscript preparation, and the decision to submit should be confirmed in the submission system.

This study was financed in part by the Coordenação de Aperfeiçoamento de pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

Declaration of competing interests

I have nothing to declare.