Teresa Cristina Monte GonçalvesInterdisciplinary Laboratory of Entomological Surveillance for Diptera and Hemiptera, Oswaldo Cruz Institute/Fiocruz
Laboratory of Entomological Biodiversity, Oswaldo Cruz Institute/Fiocruz
The study of taxonomy has used different approaches. The oldest approach is based on morphological features, also called taxonomy a. It was followed by biochemical taxonomy, or taxonomy b, and, more recently, by molecular taxonomy or taxonomy g.
Morphological taxonomy, which is based on the external morphology of the different development stages (egg, nymph and adult) and their chromatic patterns, requires previous knowledge of all structures so it is possible to identify a specimen using its only tool: the dichotomous key. However, using other techniques complements this approach and makes not only species diagnostic more correct, but also the correlation between them, also highlighting complexes of species and cryptic species.
In 1935, Galliard showed for the first time the importance of the exochorial structure to identify the different species of insects. After that, there have been various studies with morphological and morphometric approaches regarding the egg and the operculum of different species, using optic microscopy and scanning electron microscopy (Figure 1).
According to Barata, triatomine eggs are very varied in shape, ranging from almost spherical in Panstrongylus geniculatus to cylinder-shaped in Psammolestes arthuri, and in size, with an average length of 0.96 mm inAlberprosenia malheroi and 4.01 mm in Dipetalogaster maxima (Figure 2).
As for the morphological aspect, the egg has a chorial rim, and the operculum has an opercular rim. Between them is the spermatic groove, where micropyles are located and, right above them, aeropyles, smaller, yet more numerous (Figure 3).
Several studies show that according to the genus, eggs may present structures such as neck or collar and lateral flattening, as well as exochorial architecture that can vary from hexagonal infundibuliform cells to polygonal cells, in this case presenting ornamentation consisting of perforations (Figures 4, 5 and 6).
Morphology of nymphs and adults
Kissing bugs belong to the Heteroptera sub order, whose representatives have their bodies divided into head, thorax and abdomen. The head contains most of the sensitive organs, such as antennae, composite eyes and ocellus, and the mouth parts, while the chest contains locomotor appendages, legs and wings. The abdomen contains the insect’s genitalia, in its posterior portion (Figure 7).
In the Heteroptera sub order, the mouthpieces are of the piercing and sucking type, consisting of a labrum and three or four segments, which characterizes the eating habits of true bugs. In the hematophagous, it is straight, with three segments; in the predator, it is curved, with three segments; and in phytophagous, it is straight, with four segments (Figure 8). The labrum contains the mouthpieces, the serrated-edged mandibles, and the maxilae, which work as a stylus. This labrum-stylus ensemble is called a rostrum.
Among heteroptera, there are specimens that look very similar to triatomines, not only in their general appearance, but also for their color, which makes many laypeople to believe they are kissing bugs.
The development of triatiomines is of the hemimetabolic type, i.e. they have an egg phase followed by five nymph stages, all the way to adult.The young forms differ from adults by the following aspects: in the head, the rostrum does not reach the prosternum, the ocelli are absent, and the compound eyes are smaller than those of the adult forms. In the thorax, the pronotum is devoid of tubercles and carinae, and the prosternum has no stridulatory groove (Figure 9). The segmentation of the legs is identical to that of an adult, with the exception of the tarsi, which are dimers, i.e. contain two segments.
From the third stage onwards, it is already possible to visualize, in the meso and the metathorax, the wing tags, which begin to be named wing cases in the 4th and 5th stages (Figure 10).
The abdomen has 11 complete segments. The 10th and 11th segments are the anal tube and the anus, respectively. On each segment, laterally, it is possible to observe connexival spots. The 5th-stage nymphs present the 8th and 9th ventral segments modified into genital plates, whose morphology allows observers to identify the future sex of the specimen (Figure 11).
To identify the species in their young form, in 1956 Galvão created a dichotomous key for nymphs of the most common Brazilian species; Lent and Wygodzinsky, in 1979, for 1st- and 5th-stage young forms, but only at a genus level. Girón and collaborators updated the key by Lent and Wygodzinsky and included new or revalidated species and genuses.
In triatomines, the body can present variations in size, color, hairiness and in the shape of some structures, according to the species. The head, which contains the higher number of sensitive organs, contains a pair of compound eyes, a pair of ocellus, a pair of four-segment antennae and the mouthparts, in a ventral position. All these structures contain sensilla, responsible for orientation, as mentioned by Lorenzo and collaborators, and for communication, as described by Figueiras and collaborators.Other structures in the head can present different shapes, size and color, and are therefore relevant for the identification of the species. The middle portion of the head contains the followed by the clypeus and the pre-clypeus, the latter on the apex of the head. Laterally there are the genas and the jugas, whose shape and length can also vary, according to Lenta nd Wygodzinsky (Figure 12).
The compound eyes consist of hexagonal ommatidia, either regular or not, occasionally presenting hairs implanted between the ommatidea, called “erwinilae” (Figure 13), as described by Otiz and Boszko.
The ocelli are located after the compound eyes, and between them there can be a bump called post-ocular callosity, typical of the Rhodniini tribe, which includes the genuses Rhodnius and Psammolestes (Figure 14).
The antenna consists of four segments: scapus, pedicellus, the longest, and flagellum, which comprises two segments, the flagellomeres. All segments contain sensorial structures (Figure 15), classified by Catalá as mechanoreceptors, chemoreceptors, and thermohygroreceptors.
The antennae are inserted in antennal sulcus, whose location may help differentiate the three genuses that include the species of higher epidemiological significance, Rhodnius, Panstrongylus, and Triatoma. Therefore, using as a reference the distance between the anterior rim of the compound eye and the apex of the clypeus, insects which have antennal sulcus inserted before half this distance, i.e. close to the compound eye, belong to the Panstrongylus genus. Those whose sulcus is inserted right in the median line of that distance include the species of genus Triatoma, and those whose implantation is located after half this distance, i.e. on the apex of the head, are typical of genus Rhodnius (Figure 16).
The thorax is divided in three segments: prothorax, mesothorax, and metathorax. These are divided in dorsal, called notum, lateral – pleura and ventral – sternum (Figure 17).
The prothorax is the most developed segment, dorsally represented by the pronotum. This is divided into anterior and posterior lobes. The first may or may not present discal and lateral tubercles, while the latter has submedian carinae (Figure 18). Both have colors that may vary in between the different species, and are therefore relevant from the standpoint of taxnomy. The lateral region has no taxonomical relevance. Ventrally, only the prosternum has a striated structure between the first pair of coxae, the stridulatory groove, triangular in shape (Figure 19). Repetitive head movements cause friction between the apex of the rostrum and this structure, producing a sound with a very similar frequency spectrum, even though the formats are different, as are the distances between the grooves. Schilman and collaborators therefore suggested that its action is not specific, and its function is that of stopping predators.
The mesothorax (Figure 17) has its mesonotum practically covered by the pronotum; only a triangular structure (scutelum) is visible (Figure 18). The mesopleura and the mesosternum have no taxonomical relevance (Figure 20).The metathorax has a very reduced metanotum and only the metapleura and the metasternum are visible (Figure 20).
The locomotory appendages, legs and wings, are placed in the pro, meso and metathorax, and meso and metathorax, respectively (Figure 20). The different leg segments can have colors that help identify the species. The tibias can have, in their apical region, the ctenidium and the tibio-tarsal band (Figure 21).
The pro, meso and metapleura, unlike those of other insect groups, are not taxonomically relevant. Between the pro and the mesopleura, and between the meso and the metapleura are located the thoracic respiratory spiracles, while the metapleura contains the opening of the scent gland (Figure 22).
The abdomen consists of 11 dorsal segments or urotergites and 11 ventral segments or urosternites; the 10th and 11th segments correspond to the anal tube and the anus, respectively. Laterally, on both sides, are the connexives, whose function is to provide dilation of the abdomen during the blood meal. This structure is taxonomically relevant due to the variability of the color pattern between some species (Figure 23).
In females, the abdomen contains nine dorsal segments and seven ventral segments, because the 8th and 9th sternites have changed into structures called gonocoxae and gonapophysis, to form the external genitalia (Figure 24).
According to Lent and Wygodzinsky, the uniformity of these structures made their use impossible for taxonomy purposes, which is why no more studies were made with this approach.Males have seven dorsal segments and seven ventral segments. The 8th and 9th segments changed to form structures of the external genitalia. The 8th segment is a C-shaped structure that covers part of the 9th segment, also called a pigophore. The pigophore may present a pointy structure in the distal dorsal region, called pigophore median process. Within the pigophore is the copulating organ, or phallus, divided in edeagus and articular apparatus. The edeagus consists of an ellipsoid capsule that is limited ventrally by the conjunctive and dorsally by the phallosome. Within the edeagus are chitinized structures attached to each other by membranes. The articular apparatus, responsible for the movement of the edeagus during copulation, is a chitinized structure. When at rest, it is located dorsally on the edeagus (Figure 25).
According to Lent and Wygodzinsky, the different structures that make up the male genitalia can be used for taxonomy purposes, which is why they are included in the dichotomous key for species identification.
Getting to know their morphology is the first step for the taxonomical identification of a living being, including triatomines (Lent & Wygodzinsky, 1979; Galvão et al 2003; Costa et al 2013). However, cases such as cryptic species (Noireau et al. 1998; Cortez et al 2007), phenotypical variations due to speciation processes (Costa et al 2016) and phenotypical plasticity (Dias et al. 2008; Gonçalves et al 2013) require more thorough studies at a molecular level, as well as phylogenetic correlations (Hypsa et al. 2002; Monteiro et al 2004; Almeida et al. 2009; Teves at al. 2016).
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Jacenir Reis dos Santos-Mallet and Suzete Araujo Oliveira Gomes
Leishmaniasis Laboratory of the Oswaldo Cruz Institute/Fiocruz
The first studies on insects regarded only external characteristics, due to the inexistence of magnifying tools capable of allowing for the visualization of their internal organization. The first work on the internal anatomy of insects was written by Italian physician Malpighi, on silk worms. The improvement of observation tools, such as magnifying glasses, light microscopes and, more recently, electron microscopy, has made it possible to analyze and comprehend a series of important phenomena, obviously complemented with studies related to the physiology and biochemistry of cells.
The internal morphology of triatomines has been studied by countless authors, in papers describing their anatomy, histology, and ultrastructure of organs and systems. Right below the cuticle is a large amount of fat, surrounding various organs, such as the digestive tube, gonads, glands etc.
The digestive system of triatomines is divided in 3 parts: foregut, midgut, and hindgut.
It is preceded by the rostrum, which, when at rest, is adapted to the inferior part of the head and contains the set of stylos used to suck blood and inject the substances produced by the salivary glands.The pharynx is a part of the digestive tube that is highly specialized in blood sucking. It is continuous with the esophagus, all the way to the midgut (Figure 1).
Contains the largest portion of the triatomine’s digestive tube. It is formed by the promesenteric portion (stomach) and the post-mesenteric portion (intestine). When full, the stomach takes up a large part of the cavity in the insect’s body, pushing the other organs to the sides (Figures 2 and 3).
In most hematophagous insects, the midgut is lined with a peritrophic membrane or matrix that protects epithelial cells from abrasion by fragments of intestinal contents. Other functions have been described for the peritrophic membrane, including: a barrier against pathogen invasion, parasite-vector interaction site, compartmentalization of the food bolus, separation of the intestinal epithelium by the formation of the endo- and ecto-peritrophic space.
Hemiptera have a delicate pseudo-peritrophic membrane surrounded by microvilli, extending to the lumen and ending up in a cul-de-sac. It contains lipoproteins, but no chitin, and is called perimicrovillar membrane. In triatomines, the pseudoperitrophic membranes are synthetized by the epithelium of the total digestive tract, in all stages. These structures consist of glycophospholipids, proteins, carbohydrates and hydrolytic enzymes. In addition, this membrane has selective permeability, allowing digestive enzymes through so they can get to the food bolus and so digestive products can be absorbed by the intestinal epithelium.
One of the important stages in the development cycle of Trypanosoma cruzi within its invertebrate host is the interaction between the surface of the protozoan and the molecules present in the perimicrovillar membrane of the intestinal tract of triatomines.
The proctodeum, consisting of rectal ampulla and the rectum itself, forms the final part of the digestive tract and plays an important role in the reabsorption of water and minerals. The rectal ampulla consists of a muscular sac with considerable distension capacity, where the four Malpighi tubes, of different sizes, end up. The rectal ampulla contains the feces and urine which will be later eliminated through the rectum (Figures 4 and 5). In infected insects, metacyclic trypanosomes maintained in the rectal ampulla are eliminated in the feces and transmitted to the new vertebrate hosts.
Salivary glands are very diverse in terms of number, size, shape and situation in the different triatomines. In general, they are located in the thoraxic cavity, near the initial part of the digestive tube, where they have enough room to develop. Their shift to the abdominal region is a result of peristaltic movements of neighboring organs. There are three pairs of salivary glands and the nomenclature used to describe them is D1, D2 and D3 (Figure 1).Pair D1 corresponds to the main glands. D2 indicates supplementary glands. D3 refers to accessory glands. In all species of triatomines studied, all three pairs of salivary glands are always found, with the exception of genus Rhodnius, which does not possess the typical D3 glands. In general, glands D1 and D2 are milky white or yellowish, as observed in P. megistus. However, in genus Rhodnius glands D1 and D2 are elongated and red (Figure 6),
The forms of Trypanosoma rangeli that invade the salivary glands of vector insects are introduced in the vertebrate host during salivation, early in the bloodmeal.
The combined action of ectockinases and ectophosphatases may be regulating some cellular mechanisms, especially in the plasma membrane. The ectophosphatase intervenes in the adhesion of funghi to epithelial cells. More recently, Gomes and collaborators characterized, on the surface of the salivary glands of R. prolixus, acid ectophosphatase action associated to the adhesion of T. rangeli.
Malpighian tubulesThe excretory system of triatomines consists of four Malpighian tubules that end up in a large rectal sack, divided in two segments, proximal and dorsal, in permanent contact with hemolymph (Figure 7).
The primary function of Malpighian tubules is transepithelial transport, resulting in the secretion of ions and excess water, as described by Madrell. The proximal and distal portions of these tubules have simple epithelium lined with a basal layer. The cytoplasm is homogeneous in the proximal region and there are numerous vacuoli, granules and minerals such as phosphorus, chlorine, calcium, potassium, manganese, magnesium, and iron in the distal region. Mitochondria can be seen in the entire epithelium, in both regions and within microvilli (Figure 8).
The glycogen found in the Malpighian tubules is possibly the main energy reserve for the cells. The ions are used in the regulation of cellular volume, as described by Santos-Mallet. Caruso-Neves and collaborators described that Na + K–ATPase and protein kinase are enzymes associated to cell volume regulation.
In the circulatory system of insects, blood circulates in the entire body cavity, irrigating the various tissues and organs. There is a special pumping organ, called dorsal vessel, located dorsally along the insect’s body, which pumps blood from the posterior part of the body and evacuates it into the inner cavity of the head. From this cavity, blood circulates again along the dorsal part of the body, where it is removed from the head and pumped forward again, and so on. This process is called an open circulatory system, and the entire body cavity where the blood circulates is called hemocele (Figure 9).
The fluid that circulates through the insect’s body cavity is called hemolymph. It consists of a liquid part (the plasma) and a selection of free cells called hemocytes.
As the name implies, the dorsal vessel is located directly beneath the insect’s dorsal area, extending from the posterior end of the abdomen to the head. It is the main pulsatile organ responsible for blood circulation.
The dorsal vessel is divided in two parts: a posterior part, called heart, and an anterior part, called aorta. In general, the heart is the pulsatile portion, while the aorta is the tube that carries blood to the front part of the body and releases it into the head.
The heart is more or less dilated in each segment, forming segments called chambers. Each chamber has a pair of lateral openings, or ostia, through which the blood enters the chamber. The aorta is an extension of the head, of a simple tubular shape, that also pulsates and consequently functions as an accessory for the head, resulting in circulation.
Connected to the inferior side of the heart there are pairs of muscle bands known as alary muscles or aliform muscles. These muscles separate the main body cavity, called dorsal diaphragm (Figure 9), and the region around the heart, called dorsal sinus. The diaphragm and the sinus extend along the heart only, and do not develop in the aorta region.Within the heart there are the so-called pericardial cells (Figure 10).
Like in other insects, tissue oxygenation ini triatomines is the responsibility of the tracheal system, which begins with small openings on the external surface of the exoskeleton, called spiracles or respiratory stigmas, and is internally distributed to all organs by means of delicate tubes, the tracheas and tracheoles, which carry oxygen to the cells (Figure 11).
The cephalic region contains two large-caliber tracheal branches, leaving from the first pair of thoracic spiracles. In the thorax, beginning at each one of the two pairs of thoracic spiracles, there are two main trunks, going towards the anterior and posterior regions of the insect’s body. In the abdominal region, the tracheal system is more complex, beginning at each pair of abdominal spiracles, originating secondary branches that surround all the organs, as described by Lacombe (Figure 12).
The tracheas are invaginations of the tegument. They have a silvery color and a typical striated appearance, because their inner lining is formed by chitinized filaments (taenidia), usually spiral in form (Figure 13). This inner layer, consisting of kitin, is known as intima or endotrachea (Figure 14).
Female reproductive system
Consists of a pair of ovaries, a pair of lateral oviducts, a common oviduct, the bursa copulatrix, a spermatheca and the vagina.The ovaries (Figure 15), supported by a terminal filament attached at the height of the thoracic segment, are elongated and surrounded by tracheoles and tracheal bags, which increase gas exchanges. Each ovary consists of seven ovarioles, which, after a short canal, end at a single point, the chalice, which continues on with the lateral oviduct. These come together in the posterior region, to form the common ovoduct, then called bursa copulatrix and ending in the vagina (Figure 16).
One pair of accessory glands and the odd spermatheca end up in the bursa copulatrix. The function of the accessory glands is to produce mucus that will envelop the eggs, either laid free or fixated to the substrate. All species of the Rhodniini tribe and some of other genuses fixate their eggs. The spermatheca is the structure that stores the spermatozoa and ensures female fertility for some time after copulation.
The internal genitalia of males, in the adult phase, presents a pair of oval-shaped testes, whitish in color, located between the 2nd and the 5th abdominal segments, surrounded by the testicular capsule and by countless tracheas and tracheoles. The entire reproductive system is supported by the terminal filaments that are anchored at the height of the thoracic segment.
Inside the testes are the seven testicular follicles, of different sizes and arranged in an entangled fashion, which makes it possible to characterized them as indicated by Gonçalves and collaborators for the different genuses and more recently in populations of Triatoma brasiliensis by Freitas and collaborators.From each testicular follicle leaves a vas efferens that joins the medium portion of the testes, originating the vas deferens. The vas deferens crosses the membrane and follows towards the posterior region of the insect’s body. The median portion of the vas deferens dilates to form the seminal vesicle, then quickly returns to its normal caliber (Figure 17),
The distal region of the vas deferens receives the glandular canal, connected to four pairs of accessory glands (Figure 18), responsible for the production of the secretion that will encase the spermatozoa, therefore originating the spermatophore.
Almost all hemiptera have scent glands in the thoracic region. The secretion of this gland has a strong odor that has a repulsive effect, and in some cases it can even cause respiratory intoxication, as described by Carayon. Brindley refers to the presence of a pair of glands in Rhodnius prolixus, and since then these glands in triatomines are called Brindley glands.
These glands are located in the dorso-ventral region of the metathorax and have an orifice at the height of the third pair of legs. In Panstrongylus megistus this orifice is shaped like a keyhole, rounded in shape, about 1 mm of diameter and hyalin appearance, reaching the second abdominal segment.
Teresa Cristina Monte Gonçalves
Interdisciplinary Laboratory of Entomological Surveillance for Diptera and Hemiptera, Oswaldo Cruz Institute/Fiocruz
Laboratory of Entomological Biodiversity, Oswaldo Cruz Institute/Fiocruz
The development pattern of triatomines is hemimetabolic, that is, they have one egg phase, five nymph stages, and the adult stage.
The development time varies in between the different species, which justifies the fact that some have a short life cycle (less than three months), while others have a long life cycle of up to two years. In laboratory conditions, most species have an average cycle of 6 to 15 months.
However, the complementation of the biological cycle depends on abiotic factors, such as temperature, which must be between 27 and 30° C, and relative humidity, between 60 and 85%, depending on the species, as shown by Costa and collaborators and by Gonçalves and collaborators. When these conditions are very altered, morphological anomalies may occur after ecdysis, laying and eclosion of the egg.
Hematophagy occurs in all development stages of triatomines. However, cannibalism can occur, especially by younger nymphs over the more developed ones, as well as feeding on cockroaches’ hemolymph, as shown by Lorosa and collaborators. Fifth-stage nymphs ingest a larger amount of blood. In the adult phase, both females and males are hematophagous, which increases the potential transmission of Trypanosoma cruzi. Blood is extremely important for females, for the maturation of the ovaries and for the laying of the eggs.
At least one blood meal is required to make sure the insect can move on from one phase to the next, as abdominal distension and protein factors obtained from the blood meal (hemoglobin) are the factors that activate the neurosecreting cells. These cells will cause a sequence of stimuli sent to the brain, which will be responsible for producing molting (ecdysone) and growth hormones (juvenile hormone). Even during the adult phase, the growth hormone is important for egg maturation.
Defecation time is extremely important to characterize the vectorial potential of a triatomine species, as those that deposit their feces while still on the host have a higher chance of transmitting than those that defecate away from their food source. This means different species can be classified as “good” or “bad” transmitters of T. cruzi, according to Lazzari.
Resistance to fasting is a characteristic of insects in general. This is only possible thanks to the food reserves accumulated in an amorphous mass found in the hemocele, called fatty body.
In triatomines, the period of fasting resistance time is higher in 5th-stage nymphs, but it can vary in between species. The minimum and maximum periods for fifth-stage nymphs may vary from 58 to 217 days and, in adults, from 38 to 89 days, as highlighted by Cortéz and Gonçalves. This aspect of their biology is extremely important, especially when it comes to anthropophilic species or species that tend to form colonies, as it helps planning vector control and elimination programs.
This aspect of their biology is extremely important, especially when it comes to anthropophilic species or species that tend to form colonies, as it helps planning vector control and elimination programs.
Triatomine dispersion is another relevant aspect from the epidemiological standpoint. Dispersion can take place via two mechanisms: a passive one, using the vertebrate host, and an active one, when insects move by themselves.
The active dispersion of triatomines is directly associated to the nutritional state of the adult forms and to the regulation of population density. Anthropic actions on the environment contribute significantly to this dispersion, as it scares off or causes the death of hosts of wild triatomines.
The flying capacity of triatomines is a characteristic that can be progressively reduced in domestic populations, as shown by Schofield and collaborators. On the other hand, wild species maintain their flying capacity. For Triatoma infestans, the average flight distance is about 200 meters, as described by Shofield and Matthews, while in the field Schweigmann and collaborators have observed flights of more than 1 kilometer.
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