Highlights, challenges, and perspectives in basic and applied chemical ecology of triatomines.

Triatomines are vectors of Chagas disease. Due to failures in their control, there is an urgent need for more efficient and environmentally friendly monitoring and control tools. These hematophagous insects rely heavily on chemical information from the environment to detect hosts and cues/signals from conspecifics. Chemical ecology includes the elucidation of the functional role of chemicals mediating interactions between organisms. Studies on the chemical ecology of triatomines are leading to novel methods for their monitor and control. Thus, laboratory tests to develop chemical attractants and repellents are promissory and have led to the design of, for example, efficient baited traps. However, the monitoring and control tools proposed until now have not been as effective in the field.

Two sticky traps baited with synthetic host odors to capture Triatoma infestans, an important vector of Chagas disease.

Chagas disease is a vector-borne disease caused by Trypanosoma cruzi, which is transmitted by triatomine insects. Triatoma infestans is one of the main vectors. Efforts to eliminate T. infestans have often failed in the Gran Chaco, the largest endemic area of this species. Known methods for assessing triatomine house infestation include timed-manual collections by skilled personnel, bug notifications by householders' and/or non-baited detection devices. However, the detection sensitivity of those methods needs to be improved, especially when the bugs are present at low densities. In this work we design and evaluate the performance of two types of sticky traps (pitfall and climbing traps), when baited with a synthetic host odor lure, to capture T. infestans nymphs within an experimental box under semi-controlled laboratory conditions. Nine assays were conducted for each type of trap using a different experimental box per type of trap design and per treatment. These treatments were: test (T, trap baited with the synthetic lure), positive control (C+, trap baited with a mouse) and negative control (C-, empty trap). One hundred percent of the sticky pitfall and 89% of the climbing traps baited with the synthetic lure captured at least one insect. Moreover, the sticky pitfall trap and the sticky climbing trap, both baited with the synthetic lure, captured 30% and 40% of the insects in a single night, respectively. In both cases, the trap with the synthetic lure captured significantly more insects than the non-baited trap. However, the synthetic lure could be improved, as the traps with this lure captured significantly less insects than the traps with a live host. In summary, the two types of synthetically-baited traps tested were able to capture T. infestans nymphs, indicating that both designs are effective under the laboratory experimental conditions and insect abundance used in this work. These traps deserve to be tested in a field setting.

Novel repellents for the blood-sucking insects Rhodnius prolixus and Triatoma infestans, vectors of Chagas disease.

BACKGROUND: Studying the behavioral response of blood-sucking disease-vector insects to potentially repellent volatile compounds could shed light on the development of new control strategies. Volatiles released by human facial skin microbiota play different roles in the host-seeking behavior of triatomines. We assessed the repellency effect of such compounds of bacterial origin on Triatoma infestans and Rhodnius prolixus, two important vectors of Chagas disease in Latin America. METHODS: Using an exposure device, insects were presented to human odor alone (control) and in the presence of three individual test compounds (2-mercaptoethanol, dimethyl sulfide and 2-phenylethanol, the latter only tested in R. prolixus) and the gold-standard repellent NN-diethyl-3-methylbenzamide (DEET). We quantified the time the insects spent in the proximity of the host and determined if any of the compounds evaluated affected the behavior of the insects. RESULTS: We found volatiles that significantly reduced the time spent in the proximity of the host. These were 2-phenylethanol and 2-mercaptoethanol for R. prolixus, and dimethyl sulfide and 2-mercaptoethanol for T. infestans. Such an effect was also observed in both species when DEET was presented, although only at the higher doses tested. CONCLUSIONS: The new repellents modulated the behavior of two Chagas disease vectors belonging to two different triatomine tribes, and this was achieved using a dose up to three orders of magnitude lower than that needed to evoke the same effect with DEET. Future efforts in understanding the mechanism of action of repellent compounds such as 2-mercaptoethanol, as well as an assessment of their temporal and spatial repellent properties, could lead to the development of novel control strategies for these insect vectors, refractory to DEET.

An inside look at the sensory biology of triatomines.

Although kissing bugs (Triatominae: Reduviidae) are perhaps best known as vectors of Chagas disease, they are important experimental models in studies of insect sensory physiology, pioneered by the seminal studies of Wigglesworth and Gillet more than eighty years ago. Since then, many investigations have revealed that the thermal, hygric, visual and olfactory senses play critical roles in the orientation of these blood-sucking insects towards hosts. Here we review the current knowledge about the role of these sensory systems, focussing on relevant stimuli, sensory structures, receptor physiology and the molecular players involved in the complex and cryptic behavioural repertoire of these nocturnal insects. Odours are particularly relevant, as they are involved in host search and are used for sexual, aggregation and alarm communication. Tastants are critical for a proper recognition of hosts, food and conspecifics. Heat and relative humidity mediate orientation towards hosts and are also important for the selection of resting places. Vision, which mediates negative phototaxis and flight dispersion, is also critical for modulating shelter use and mediating escape responses. The molecular bases underlying the detection of sensory stimuli started to be uncovered by means of functional genetics due to both the recent publication of the genome sequence of Rhodnius prolixus and the availability of modern genome editing techniques.

Herbivore Damage and Prior Egg Deposition on Host Plants Influence the Oviposition of the Generalist Moth Trichoplusia ni (Lepidoptera: Noctuidae).

Female insects have the difficult task of locating host plants that maximize the survival and success of their offspring. In this study, the oviposition preferences of the cabbage looper moth, Trichoplusia ni (Hübner), for soybean plants, Glycine max (L.), under various treatments-undamaged, mechanically damaged, damaged by T. ni or Spodoptera frugiperda (Smith) larvae or by Bemisia tabaci (Gennadius) adults, egg-free plants, and plants previously oviposited by conspecific or heterospecific females (S. frugiperda)-were investigated using two-choice tests. Additionally, the volatile compounds emitted by the plants under the different treatments were identified by gas chromatography-mass spectrometry. Our results showed that females showed no preferences for undamaged or mechanically damaged plants. However, they oviposited more often on undamaged plants than on those previously damaged by T. ni, S. frugiperda, or B. tabaci. In contrast, females preferred to oviposit on plants previously oviposited by conspecific and heterospecific females than on egg-free plants. Plants damaged by conspecific or heterospecific larvae emitted methyl salicylate, indole, and octyl butyrate, compounds not released by undamaged or mechanically damaged plants. Whitefly damage induced the release of higher quantities of Z(3)-hexenyl acetate, (R)-(+)-limonene, and (E)-ß-ocimene compared to plants damaged by larvae and suppressed the emission of linalool. Egg deposition by conspecific and heterospecific moths induced the emission of (R)-(+)-limonene, octyl butyrate, and geranyl acetone but suppressed the release of linalool. This study showed that a generalist moth species can discriminate between plants of different quality, and suggests that females use volatile compounds as cues during this process.

Neuroethology of Olfactory-Guided Behavior and Its Potential Application in the Control of Harmful Insects.

Harmful insects include pests of crops and storage goods, and vectors of human and animal diseases. Throughout their history, humans have been fighting them using diverse methods. The fairly recent development of synthetic chemical insecticides promised efficient crop and health protection at a relatively low cost. However, the negative effects of those insecticides on human health and the environment, as well as the development of insect resistance, have been fueling the search for alternative control tools. New and promising alternative methods to fight harmful insects include the manipulation of their behavior using synthetic versions of "semiochemicals", which are natural volatile and non-volatile substances involved in the intra- and/or inter-specific communication between organisms. Synthetic semiochemicals can be used as trap baits to monitor the presence of insects, so that insecticide spraying can be planned rationally (i.e., only when and where insects are actually present). Other methods that use semiochemicals include insect annihilation by mass trapping, attract-and- kill techniques, behavioral disruption, and the use of repellents. In the last decades many investigations focused on the neural bases of insect's responses to semiochemicals. Those studies help understand how the olfactory system detects and processes information about odors, which could lead to the design of efficient control tools, including odor baits, repellents or ways to confound insects. Here we review our current knowledge about the neural mechanisms controlling olfactory responses to semiochemicals in harmful insects. We also discuss how this neuroethology approach can be used to design or improve pest/vector management strategies.

Antennal phenotype of Mexican haplogroups of the Triatoma dimidiata complex, vectors of Chagas disease.

Triatoma dimidiata (Latreille) is a species complex that spans North, Central, and South America and which is a key vector of all known discrete typing units (DTU) of Trypanosoma cruzi, the etiologic agent of Chagas disease. Morphological and genetic studies indicate that T. dimidiata is a species complex with three principal haplogroups (hg) in Mexico. Different markers and traits are still inconclusive regarding if other morphological differentiation may indicate probable behavioral and vectorial divergences within this complex. In this paper we compared the antennae of three Mexican haplogroups (previously verified by molecular markers ND4 and ITS-2) and discussed possible relationships with their capacity to disperse and colonized new habitats. The abundance of each type of sensillum (bristles, basiconics, thick- and thin-walled trichoids) on the antennae of the three haplogroups, were measured under light microscopy and compared using Kruskal-Wallis non-parametric and multivariate non-parametric analyses. Discriminant analyses indicate significant differences among the antennal phenotype of haplogroups either for adults and some nymphal stages, indicating consistency of the character to analyze intraspecific variability within the complex. The present study shows that the adult antennal pedicel of the T. dimidiata complex have abundant chemosensory sensilla, according with good capacity for dispersal and invasion of different habitats also related to their high capacity to adapt to conserved as well as modified habitats. However, the numerical differences among the haplogroups are suggesting variations in that capacity. The results here presented support the evidence of T. dimidiata as a species complex but show females and males in a different way. Given the close link between the bug's sensory system and its habitat and host-seeking behavior, AP characterization could be useful to complement genetic, neurological and ethological studies of the closely related Dimidiata Complex haplogroups for a better knowledge of their vectorial capacity and a more robust species differentiation.

Oviposition in the blood-sucking insect Rhodnius prolixus is modulated by host odors.

BACKGROUND: Triatomine bugs are blood-sucking insects, vectors of Chagas disease. Despite their importance, their oviposition behavior has received relatively little attention. Some triatomines including Rhodnius prolixus stick their eggs to a substrate. It is known that mechanical cues stimulate oviposition in this species. However, it is not clear if chemical signals play a role in this behavior. We studied the role of host cues, including host odor, in the oviposition behavior of the triatomine R. prolixus. METHODS: Tests were carried out in an experimental arena and stimuli consisted of a mouse or hen feathers. The number of eggs laid and the position of those eggs with respect to the stimulus source were recorded. Data were analyzed using the Mann-Whitney and Kruskal-Wallis tests. RESULTS: Both a mouse and hen feathers stimulated oviposition. In addition, hen feathers evoked a particular spatial distribution of eggs that was not observed in the case of mouse. CONCLUSIONS: We propose that volatile chemical cues from the host play a role in the oviposition behavior of triatomines that stick their eggs. Thus, host odor would stimulate and spatially guide oviposition.

Effects of starvation on the olfactory responses of the blood-sucking bug Rhodnius prolixus.

Blood-sucking insects use olfactory cues in a variety of behavioral contexts, including host-seeking and aggregation. In triatomines, which are obligated blood-feeders, it has been shown that the response to CO2, a host-associated olfactory cue used almost universally by blood-sucking insects, is modulated by hunger. Host-finding is a particularly dangerous task for these insects, as their hosts are also their potential predators. Here we investigated whether olfactory responses to host-derived volatiles other than CO2 (nonanal, α-pinene and (-)-limonene), attractive odorant mixtures (yeast volatiles), and aggregation pheromones (present in feces) are also modulated by starvation in the blood-sucking bug Rhodnius prolixus. For this, the responses of both non-starved and starved insects were individually tested at the beginning of the scotophase using a dual-choice "T-shaped" olfactometer, in which one of its arms presented odor-laden air and the other arm presented odorless air. We found that the response of non-starved insects toward host-odorants and odorant mixtures was odor-dependent: insects preferred the odor-laden arm of the maze when tested with α-pinene, the odorless arm of the maze when tested with (-)-limonene, and distributed at random when tested with yeast volatiles or nonanal. In contrast, starved insects significantly preferred the odor-laden arm of the maze when tested with host-odorants or yeast volatiles. When tested with aggregation be, while starved insects preferred the odorless arm of the maze; insects that were even more starved (8-9 weeks post-ecdysis) significantly preferred the odor-laden arm of the maze. We postulate that this odor- and starvation-dependent modulation of sensory responses has a high adaptive value, as it minimizes the costs and risks associated with the associated behaviors. The possible physiological mechanisms underlying these modulatory effects are discussed.

Feeding and defecation behavior of Triatoma rubida (Uhler, 1894) (Hemiptera: Reduviidae) under laboratory conditions, and its potential role as a vector of Chagas disease in Arizona, USA.

Chagas disease is caused by the parasite Trypanosoma cruzi, which is transmitted to humans by blood-sucking triatomine insects. This disease is endemic throughout Mexico and Central and South America, but only a few autochthonous cases have been reported in the United States, despite the fact that infected insects readily invade houses and feed on humans. Competent vectors defecate during or shortly after feeding so that infective feces contact the host. We thus studied the feeding and defecation behaviors of the prevalent species in southern Arizona, Triatoma rubida. We found that whereas defecation during feeding was frequent in females (93%), it was very rare in immature stages (3%), and absent in males. Furthermore, more than half of the immature insects that exhibited multiple feeding bouts (62%) defecated during interruptions of feeding, i.e., while likely on or near the host. These results indicate that T. rubida potentially could transmit T. cruzi to humans.

Infection of kissing bugs with Trypanosoma cruzi, Tucson, Arizona, USA.

Triatomine insects (Hemiptera: Reduviidae), commonly known as kissing bugs, are a potential health problem in the southwestern United States as possible vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Although this disease has been traditionally restricted to Latin America, a small number of vector-transmitted autochthonous US cases have been reported. Because triatomine bugs and infected mammalian reservoirs are plentiful in southern Arizona, we collected triatomines inside or around human houses in Tucson and analyzed the insects using molecular techniques to determine whether they were infected with T. cruzi. We found that 41.5% of collected bugs (n = 164) were infected with T. cruzi, and that 63% of the collection sites (n = 22) yielded >or=1 infected specimens. Although many factors may contribute to the lack of reported cases in Arizona, these results indicate that the risk for infection in this region may be higher than previously thought.

Host-seeking: How triatomines acquire and make use of information to find blood.

The evolution of triatomine bugs towards haematophagy has demanded different types of adaptations, i.e., morphological, physiological and behavioural. In fact, haematophagy evolved as a secondary adaptation facilitated by frequent vertebrate contact. As derived from other Heteroptera, probably from an entomophagous group, some main morphological pre-adaptations, as piercing mouthparts and sucking pumps were already present. Thus, the most important novel acquisitions of triatomines include physiological and behavioural traits to obtain and handle the blood meal. In this review, we discuss how the sensory system and the behaviour of triatomines have been shaped by natural selection to accomplish the tasks of finding a vertebrate host and getting access to its blood. The feeding behaviour of triatomines is presented in its spatial and temporal context. Finally, some methods to study these topics are described.

Roles and effects of environmental carbon dioxide in insect life.

Carbon dioxide (CO(2)) is a ubiquitous sensory cue that plays multiple roles in insect behavior. In recent years understanding of the well-known role of CO(2) in foraging by hematophagous insects (e.g., mosquitoes) has grown, and research on the roles of CO(2) cues in the foraging and oviposition behavior of phytophagous insects and in behavior of social insects has stimulated interest in this area of insect sensory biology. This review considers those advances, as well as some of the mechanistic bases of the modulation of behavior by CO(2) and important progress in our understanding of the detection and CNS processing of CO(2) information in insects. Finally, this review briefly addresses how the ongoing increase in atmospheric CO(2) levels may affect insect life.

Floral CO2 reveals flower profitability to moths.

The hawkmoth Manduca sexta (Lepidoptera: Sphingidae), an experimentally favorable Lepidopteran that is highly sensitive to carbon dioxide (CO2), feeds on the nectar of a range of flowering plants, such as Datura wrightii (Solanaceae). Newly opened Datura flowers give off dramatically elevated levels of CO2 and offer ample nectar. Thus, floral CO2 emission could indicate food-source profitability. This study documents that foraging Manduca moths prefer surrogate flowers that emit high levels of CO2, characteristic of newly opened Datura flowers. We show for the first time that CO2 may play an important role in the foraging behavior of nectar-feeding insects.

Floral CO(2) emission may indicate food abundance to nectar-feeding moths.

As part of a study of the roles of the sensory subsystem devoted to CO(2) in the nectar-feeding moth Manduca sexta, we investigated CO(2) release and nectar secretion by flowers of Datura wrightii, a preferred hostplant of Manduca. Datura flowers open at dusk and wilt by the following noon. During the first hours after dusk, when Manduca feeds, the flowers produce considerable amounts of nectar and emit levels of CO(2) that should be detectable by moths nearby. By midnight, however, both nectar secretion and CO(2) release decrease significantly. Because nectar production requires high metabolic activity, high floral CO(2) emission may indicate food abundance to the moths. We suggest that hovering moths could use the florally emitted CO(2) to help them assess the nectar content before attempting to feed in order to improve their foraging efficiency.

Sensory processing of ambient CO2 information in the brain of the moth Manduca sexta.

Insects use information about CO2 to perform vital tasks such as locating food sources. In certain moths, CO2 is involved in oviposition behavior. The labial palps of adult moths that feed as adults have a pit organ containing sensory receptor cells that project into the antennal lobes, the sites of primary processing of olfactory information in the brain. In the moth Manduca sexta and certain other species of Lepidoptera, these receptor cells in the labial-palp pit organ have been shown to be tuned to CO2, and their axons project to a single, identified glomerulus in the antennal lobe, the labial-palp pit organ glomerulus. At present, however, nothing is known about the function of this glomerulus or how CO2 information is processed centrally. We used intracellular recording and staining to reveal projection (output) neurons in the antennal lobes that respond to CO2 and innervate the labial-palp pit organ glomerulus. Our results demonstrate that this glomerulus is the site of first-order processing of sensory information about ambient CO2. We found three functional types of CO2-responsive neurons (with their cell bodies in the antennal lobe or the protocerebrum) that provide output from the antennal lobe to higher centers in the brain. Some physiological characteristics of those neurons are described.

Ultrastructure and receptor cell responses of the antennal grooved peg sensilla of Triatoma infestans (Hemiptera: Reduviidae).

Ultrastructural examination of grooved-peg (GP) sensilla on the antenna of fifth instar Triatoma infestans nymphs by scanning electron microscopy and transmission electron microscopy reveal that they are 8-18 microm long with a diameter of about 2-2.8 microm at the non-articulated base. Some pegs have a terminal pore. These double-walled wall-pore (dw-wp) sensilla have an outer cuticular wall with 13-18 longitudinal grooves at the distal part of the peg. Groove channels are present at the bottom of the grooves from which radial spoke channels lead into the inner sensillum-lymph cavity. A dendrite sheath connects the tip of the thecogen cell to the inner cuticular wall thus forming separated outer and inner sensillum-lymph cavities. Four or five bipolar receptor cells are ensheathed successively within the GP sensilla by the thecogen cell, trichogen and tormogen cells. The inner dendritic segments of each sensory cell give rise at the ciliary constriction to an unbranched outer dendritic segment which can reach the tip of the sensillum. Electrophysiological recordings from the GP sensilla indicate that they house NH3, short-chain carboxylic acid and short-chain aliphatic amine receptor cells and can be divided into three functional sub-types (GP 1-3). All GP sensilla carry a receptor cell excited by aliphatic amines, such as isobutylamine, a compound associated with vertebrate odour. GP type 1 and 2 sensilla house, in addition, an NH3-excited cell whereas the type 2 sensilla also contains a short-chain carboxylic acid receptor. No cell particularly sensitive to either NH3 or carboxylic acids was found in the grooved-peg type 3 sensilla. GP types 1, 2 and 3 represent ca. 36, 10 and 43% of the GP sensilla, respectively, whereas the remaining 11% contain receptor cells that manifest normal spontaneous activity but do not respond to any of the afore mentioned stimuli.