Laboratory culture of Triatoma sanguisuga (Hemiptera: Reduviidae) by supplementation with Drosophila artificial hemolymph media

Abstract

Triatoma sanguisuga (LeConte, 1855), the eastern bloodsucking conenose, is an epidemiologically significant triatomine vector in the United States in that it is implicated in documented autochthonous human Chagas disease cases in the United States and can be found across a large geographic area. Despite decades of research interest, this species has proven remarkably challenging to maintain in laboratory culture, with consistent developmental arrest occurring prior to the fifth nymphal instar stage when maintained on standard blood-only feeding protocols. Here, we report a successful protocol for maintaining T. sanguisuga through complete development in laboratory culture. Adult specimens were collected from Des Allemands, Louisiana, and maintained under standard laboratory conditions with blood feeding supplemented by Drosophila artificial hemolymph media (AHL). Early nymphal development proceeded through the third instar on blood meals alone, but development consistently stalled at the fourth instar. Following initiation of AHL supplementation, the first successful fourth-to-fifth instar molt was observed approximately 3 wk later, with subsequent emergence of adult insects occurring within 17 wk. The AHL supplement was readily consumed by nymphs when offered ad libitum at room temperature. This development overcomes a significant barrier to T. sanguisuga laboratory culture and provides compelling evidence for complex nutritional requirements in triatomines beyond those met by vertebrate blood alone. Laboratory-reared T. sanguisuga cultures will enable critical research on vector competence, feeding behavior, reproductive biology, and control measures essential for understanding and managing Chagas disease transmission in North America.

Introduction

Triatoma sanguisuga (LeConte, 1855), the eastern bloodsucking conenose, stands as a highly significant triatomine vector in the United States. This species is the most widely geographically distributed among this group of vectors and has a demonstrated propensity for feeding on humans and has been associated with autochthonous human Chagas disease transmission (Dorn et al. 2007, Klotz et al. 2014, Wozniak et al. 2015, Dye-Braumuller et al. 2020, Jameson et al. 2022, Peterson et al. 2024).

Despite decades of research interest, T. sanguisuga has proven remarkably challenging to maintain in laboratory culture through complete development. Early attempts to rear this species were described as being accomplished “with great difficulty,” with researchers consistently encountering high mortality rates and developmental arrest (Grundemann 1947). While other medically important triatomine species, particularly Rhodnius prolixus (Stål, 1859) and Triatoma infestans (Klug, 1834), have been successfully maintained in laboratory cultures for decades using established protocols (Wood 1954, Gomez Nunez 1964, Sutcliffe and Dotson 2024), and laboratory maintenance of the more geographically related Triatoma rubida (Uhler, 1894) and Triatoma gerstaeckeri (Stål, 1859) have recently been described (Adhikari 2025), T. sanguisuga has remained recalcitrant to standard rearing protocols.

Growing evidence indicates that triatomines, particularly nymphs, actively seek and consume non-vertebrate blood food sources in natural settings with sugar feeding behavior documented in multiple triatomine species (Díaz-Albiter et al. 2016, Costa et al. 2024). Additionally, the phenomenon of hemolymphagy—the feeding of triatomine nymphs on hemolymph from arthropod hosts—has been observed both in laboratory settings and inferred from field observations (Ruas-Neto et al. 2001, Freitas et al. 2005, Alves et al. 2011, Beatty et al. 2025).

The ecological and evolutionary context of triatomine feeding behavior provides important insights into potential nutritional requirements. Recent molecular evidence has revealed the presence of plant DNA in field-collected triatomines and the identification of amylase enzymes in triatomine genomes, suggesting an evolutionary capacity for carbohydrate utilization that extends beyond the traditional understanding of these insects as obligate hematophages (Díaz-Albiter et al. 2016, Da Lage et al. 2024). This evidence supports the hypothesis that triatomines may be in the process of evolving fully hematophagous behavior and may retain ancestral nutritional requirements for carbohydrate-containing food sources.

Building on these insights into triatomine nutritional ecology, we hypothesized that the developmental challenges observed in laboratory-reared T. sanguisuga might result from the absence of specific nutrients normally obtained through hemolymphagy in natural settings. Rather than establishing complex co-culture systems with arthropod prey species, we investigated whether Drosophila artificial hemolymph media could provide the necessary nutritional supplementation to overcome the observed developmental challenges.

Here, we present a successful protocol for maintaining T. sanguisuga through complete development in laboratory culture. Our approach combines traditional blood feeding with supplementation using Drosophila artificial hemolymph media, resulting in successful molting through all nymphal stages and the production of reproductively fit adult insects. This development provides a practical tool for T. sanguisuga research, supports the emerging understanding of triatomine nutritional requirements and the evolutionary context of hematophagous behavior in this medically important group of insects, and provides a starting point for controlled investigations into the nutritional requirements of this species.

Materials and Methods

Insects

Adult T. sanguisuga specimens were collected during summer 2024 from Des Allemands, Louisiana (29.8°N, 90.4°W) as previously described (Vazquez-Prokopec et al. 2004, Rebollar-Téllez et al. 2009). Three male and 3 female adults were selected for the establishment of cultures. All collected specimens tested positive for Trypanosoma cruzi infection via microscopic examination of fecal samples, consistent with the high infection rates previously reported for this population (Cesa et al. 2011, Dumonteil et al. 2024).

Insects were housed following standard protocols (Sutcliffe and Dotson 2024) with minor revisions. Insects were maintained in rearing containers consisting of 480 ml clear plastic containers with mesh lids. Folded seed germination paper was provided as a resting substrate. Rearing containers were maintained at 28 ± 2 °C and 80 ± 5% relative humidity in an environmental chamber under a 12:12 light:dark photoperiod. The parental generation was housed collectively, and all eggs produced by this cohort were maintained together as a single population.

Blood and Blood Feeding

Defibrinated bovine blood was obtained from HemoStat Laboratories (Dixon, California). Blood was aliquoted and frozen at –20 °C until use.

To feed, warmed blood was added to a glass water-jacketed membrane feeding device (Lillie Glass, Atlanta, Georgia) covered with a Parafilm M membrane as described previously (Rutledge et al. 1964). Water maintained at 37 °C was circulated through the system to warm the blood. Insects were fed every 7 to 14 d and allowed to feed to repletion or for a maximum of 4 h.

Drosophila Artificial Hemolymph Media and Feeding

Drosophila artificial hemolymph media (AHL) was prepared as in a previously published protocol (Freyberg 2025). Briefly, stock solutions of the constituent solutions were prepared as 0.2 µm-filtered solutions. Working solution was prepared in a sterile cabinet following the protocol recommendations and stored at 4 °C until use. The working solution contained 108 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 8.2 mM MgCl₂, 1 mM NaH₂PO₄, 10 mM sucrose, 5 mM trehalose, 5 mM HEPES, 4 mM NaHCO₃ in a 100 ml volume of deionized water, pH = 7.5. Working solutions were used directly out of 4 °C storage and allowed to come to ambient temperature while in the cage.

Two months after fourth instars were visible in the rearing container, 3 to 4 ml room temperature AHL was offered to the cage through a Parafilm M membrane. This meal was left for the insects to feed ad libitum between blood feeds. A small amount of Spirulina powder in tap water was added above the AHL for visual contrast (Fig. 1). The feeding schedule with AHL consisted of 6 d of ad libitum AHL, starvation overnight, followed by blood feeding on day 7. AHL was replaced during the week if it became turbid or the membrane ruptured, but ingestion of AHL was not recorded or monitored.

Fig. 1.

Blood feeding (left) and AHL supplementation (right) setup utilized in this study. Left: A blown glass feeding device is fitted with a Parafilm M membrane, and the inner reservoir is filled with defibrinated bovine blood. A water bath, maintained at 37 °C, and a pond pump circulate warm water through the outer jacket of the feeding device. Right: A small plastic cup is fitted with a Parafilm M membrane, and AHL is pipetted between the membrane and the cup and sealed such that a small bubble of AHL is maintained on the underside of the cup. A small amount of Spirulina powder is mixed with water and added to the interior of the cup to provide visual contrast for the insects.

Developmental Monitoring and Data Collection

Population counts were conducted during routine maintenance and cage cleaning events throughout the study period. Prior to the introduction of AHL supplementation, general population counts were recorded without detailed developmental staging, though general impressions were noted. Following the initiation of supplementation, weekly counts by developmental stage were conducted to track developmental progress. In all cases, cage cleaning happened only on an as-needed basis, and seed germination paper containing excreta was included in all cleaned cages.

Data Analysis

Population counts and developmental stage distributions were recorded in spreadsheet format and analyzed using descriptive statistics. As this was a preliminary observational study of a culture protocol of T. sanguisuga in a defined system, and individual insects were not followed over time, statistical analyses of developmental timelines compared to control groups were not feasible.

Results

All founding adults readily adapted to laboratory conditions and began producing viable eggs within one month of collection. Mating behavior was observed regularly, and eggs were deposited individually throughout the container.

First, second, and third instar nymphs were successfully located and fed from the membrane feeding apparatus, and development proceeded without detailed observations. Consistent with our previous rearing attempts, development eventually plateaued with the population consisting predominantly of third instar individuals that had been in that stage for 8 or more weeks without development. This was determined by dated, routine cage census counts without corresponding deaths, thus indicating that a number of individual insects were being maintained at the third instar for an extended duration. AHL supplementation was initiated two months after the first fourth instar individuals were noted.

The AHL was readily accepted by the nymphs, with feeding behavior observed within 24 h of initial presentation. Though activity metrics were not obtained before and during AHL supplementation, anecdotally, the insects supplemented with AHL were much faster and more responsive to handling than this species when maintained only on vertebrate blood.

The first successful fourth-to-fifth instar molt was observed approximately three weeks after initiating AHL supplementation. This individual was easily distinguished from third instars by its larger size, more pronounced wing pad development, and slightly altered coloration pattern. Additional fifth instar individuals appeared regularly over the subsequent weeks. The progression from fourth to fifth instar was not synchronous, with molts occurring primarily over a period of 14 wk (Fig. 2). This is represented by the opposite slopes of the count data from week 6 to week 20.

Fig. 2.

Weekly counts of T. sanguisuga culture beginning with the appearance of the first fifth instar individual. No first instars were counted over the course of data collection. Data for second instars removed for clarity. A full data table of counts are provided as Supplemental Data. Week 0 denotes the beginning of AHL supplementation.

The first adult T. sanguisuga emerged approximately 17 wk after initiating hemolymph supplementation, representing successful completion of the entire nymphal developmental sequence under laboratory conditions (Fig. 3). This individual, a male, displayed normal adult morphology, including fully developed wings, mature coloration patterns, and appropriate body proportions. Individuals in the culture continued to develop asynchronously and have yielded 6 viable adults (3 male, 3 female), and 3 failed imaginal molts thus far. The adults have been observed mating and have begun laying viable eggs (data not presented here).

Fig. 3.

Imaginal molt of T. sanguisuga reared from laboratory-deposited egg—premolt (left), ecdysis (center) and postmolt (right). During premolt, orange banding on the abdomen dulls, and pink begins to show through the venter. Ecdysis occurs over approximately 30 min, and newly emerged adults slowly achieve their characteristic coloration within 24 h. A timelapse video of the imaginal molt of T. sanguisuga is provided as Supplemental Data.

Discussion

The development of a protocol allowing for the successful development of T. sanguisuga in the laboratory represents a significant advancement in triatomine vector biology research. This species has proven difficult to maintain in culture, which makes evaluation of their behavior and physiology in laboratory settings challenging (Grundemann 1947, Moore 2017). This vector has been implicated with locally acquired human cases of Chagas disease in the southeastern United States, with natural infections documented across the southeastern United States (Jameson et al. 2022), yet much remains unknown about their association with humans and domestic animals due to laboratory cultivation challenges.

Our findings suggest that the addition of AHL as a dietary supplement successfully overcomes a poorly characterized developmental barrier that has historically prevented T. sanguisuga laboratory culture. The success of our approach aligns with a growing body of literature suggesting that some species of triatomines retain significant nutritional requirements beyond those met by vertebrate blood alone. Recent evidence documenting sugar feeding behavior in multiple triatomine species, particularly in nymphal stages, supports the concept that these insects have not yet evolved complete dependence on vertebrate blood sources (Díaz-Albiter et al. 2016, Costa et al. 2024). This complexity in the nutritional ecology of triatomines may be an underappreciated factor in laboratory settings.

While there is no direct justification for the specific use of AHL beyond standardized protocols and convenience, the natural occurrence of hemolymphagy in triatomines provides context for understanding why artificial hemolymph media may have proven effective in our study (Durán et al. 2016). AHL also includes several components that are likely crucial for triatomine development. The high sugar content, including trehalose, sucrose, and glucose, provides readily available energy sources that may be essential for the energy-intensive process of molting. Trehalose, in particular, has been shown to be well-tolerated by triatomines and may serve as an important energy storage molecule (Costa et al. 2024). Future studies examining the role of the various components of AHL may provide more detailed insights into the specific nutritional requirements for T. sanguisuga development.

Should AHL supplementation be able to sustain a robust colony over the coming years, the laboratory-reared insects will enable controlled studies of vector competence for different T. cruzi strains, feeding preferences and host-seeking behavior, responses to environmental variables, and susceptibility to insecticidal control measures. Known-age cohorts of T. sanguisuga will be particularly valuable for studies requiring precise developmental staging or temporal control.

While our study successfully demonstrates a working protocol for T. sanguisuga culture, several important questions remain for future investigation. The specific components of the hemolymph media that are essential for successful development have not been determined, and optimization studies may simplify the protocol or reduce costs. Initial studies will focus on the introduction of AHL supplementation in the first instar to assess the growth and development metrics.

The long-term stability and genetic health of laboratory cultures established using this protocol will require monitoring over multiple generations. Potential concerns include inbreeding depression, loss of vector competence, or changes in feeding behavior that might affect the relevance of laboratory findings to wild populations. Important metrics were not recorded in this preliminary study, including mortality rates before and during AHL supplementation, and no control group of the standard rearing process was maintained in parallel. Future studies will focus in these areas.

Comparative studies examining the nutritional requirements of other difficult-to-culture triatomine species could reveal whether the principles underlying our successful approach can be more broadly applied. Such studies might also provide insights into the evolutionary relationships between different species and their degrees of adaptation to hematophagous lifestyles.

Conclusions

The development of a successful laboratory culture protocol for T. sanguisuga represents both a practical advancement for vector biology research and a contribution to our theoretical understanding of triatomine evolution and nutritional ecology. The requirement for hemolymph supplementation supports the hypothesis that triatomines are continuing to evolve hematophagous traits and retain ancestral nutritional requirements that are not met by vertebrate blood alone.

Author Contributions

Samuel B Jameson (Conceptualization [lead], Data curation [lead], Formal analysis [lead], Investigation [lead], Methodology [lead], Writing—original draft [lead]), Rachel Clear (Conceptualization [supporting], Methodology [supporting], Project administration [supporting], Writing—review & editing [supporting]), and Dawn M. Wesson (Conceptualization [supporting], Supervision [lead], Writing—review & editing [supporting])

Supplementary Material

Supplementary material is available at Journal of Medical Entomology online.

Funding

None declared.

Conflicts of Interest

None declared.