The insect vector of Chagas disease, Rhodnius prolixus, has become a very popular model organism for exploring, among other things, the physiology of insects. Its ability to remain in a state of stasis until after engorging a blood meal has focussed most studies on those physiological and developmental processes triggered by the blood meal leaving the details of its sexual physiology vague. This chapter summarizes the relationship between the male and female by describing their respective reproductive systems and genitalia, and how they function during and after copulation. A number of novel processes are noted, such as the transfer of male secretions without the formation of a spermatophore, pump/valve mechanism in the male aedeagus, sensory and a chemical means by which copulation may be facilitated, and the possible mechanism by which adhesive protein is applied to an egg during ovipositioning. Combined with knowledge of its genome, further studies into the functional anatomy of reproduction in this insect have the potential to increase our understanding of sexual reproduction in Reduviidae bugs, and to suggest new ways to control their population growth and the spread of Chagas disease.
- Rhodnius prolixus
- sexual physiology
- male genitalia
- female genitalia
- Chagas disease
- accessory reproductive glands
This chapter describes the anatomy and physiology of internal and external reproductive structures in Reduviidae bugs, the blood‐feeding insect vectors of Chagas disease. Chagas disease is endemic to Central and South America, and is also known as American trypanosomiasis . The disease is caused by a protozoan parasite,
The causative agent of Chagas disease and its mode of transmission by Reduviidae bugs was discovered by Carlos Chagas in 1909 . Although transmitted by several species of Reduviidae, one species,
In his studies on insect physiology, for which he was knighted in 1964, Wigglesworth concentrated on growth, development and metamorphosis in
2. Overall design of the adult abdomen
The adult abdomen in
3. The female reproductive system
3.1. The dorsal and ventral genital segments of the female
The genitalia of the female are attached to abdominal segment seven, and are equipped with a single dorsal sclerite and a pair of ventral sclerites (see Figure 2 ). When pulled in towards the rear of the animal, these genital sclerites cover the sclerites that surround the opening to the genital chamber. The genital chamber in
The dorsal genital sclerite is hinged on the posterior edge of full‐size abdominal segment seven ( Figure 2A ), and narrows towards its posterior tip to take on a triangular shape. In its retracted position, it sits under the animal extending ventro‐anteriorly ( Figure 2B ). It has symmetrically arranged lateral flaps ( Figure 2A ) to which the male can attach his parameres when this genital sclerite is extended during copulation. Its posterior tip has a prominent medial ridge that overlaps sclerites ventral to it when the vulva, the external opening to the vagina, is closed. When the genital segments are relaxed as a result of decapitation of the female, the third valvula becomes visible ( Figure 2C ). A slender branch of cuticle connects the lateral edge of the dorsal genital sclerite to the base of the second valvula which is one of the three pairs of sclerites associated with the vulva.
On its interior side, the dorsal genital sclerite has a pair of apodemes with each member of the pair located between the midline and the right or left side of the sclerite. Anchored to these apodemes are a pair of bilaterally symmetrical muscle bundles which fan out a short distance anteriorly to attach to the posterior lining of the vagina. Contractions of these muscles pull the dorsal genital sclerite anteriorly onto the underside of the animal to close off the vulva and the anus. Relaxation of these muscles allows the dorsal genital sclerite to extend exposing the anus during defaecation, or the vulva during copulation, egg‐laying or the expulsion of the male secretions after copulation.
While the dorsal genital sclerite covers the dorsal to lateral sides of the rear of the abdomen, the ventral to lateral sides are covered by a pair of ventral genital sclerites. The relationship of these ventral sclerites to the ventral side of abdominal segment 7 is governed by the shape of the abdomen in cross section. Whereas the dorsal abdominal surface is flat, the ventral portion forms a deep trough. The anterior part of each pair of ventral genital sclerites sits in this trough so that they lay over part of the interior side of abdominal segment 7. The eighth abdominal spiracle is located on the ventral genital sclerite, but not on its outer nor inner surface. Instead, it sits approximately midway along its lateral edge (see Figure 3 ), and this edge becomes exposed to the outside when the ventral genital segments are extended out of abdominal segment 7 to open the vagina. The ventral genital segment is attached to the inside of the ventral side of abdominal segment 7 by at least four sets of skeletal muscles. These muscles, which have yet to be fully documented, provide the female with considerable control of the sclerites of the ventral genital segment, a control that would be exercised during copulation and ovipositioning.
On the side facing the vagina, the ventral genital sclerites are directly attached to the bulk of the muscles that overlie the vagina ( Figure 3 ). The muscle bundles fan out in three different directions and become intertwined as they proceed over the vagina. The most anteriorly attached muscle bundles extend anteriorly along the ipsilateral side of the vagina, past the common oviduct, to attach to the posterior medial edge of abdominal segment 7 where the muscle bundles associated with the dorsal genital sclerite also attach. The muscle bundles attached to the mid anterior region of the ventral genital sclerite form a distinct twisting pattern, and extend directly across the body over the posterior end of the vagina to the contralateral ventral genital sclerite. The more posteriorly attached muscle bundle extends anteriorly and contralaterally travelling across the top of the vagina around the contralateral side of the common oviduct to attach to the medial posterior edge of abdominal segment 7. The interwoven nature of the muscle fibres and the diagonal pattern assumed by many of them would help to ensure that pressure generated during their contractions would be evenly spread over an exiting egg.
3.2. The vulva
The vagina opens to the outside through the vulva, which is surrounded by three sets of sclerites (see Figure 4 ). These consist of a single dorsal sclerite, a pair of lateral sclerites which are attached to the dorsal genital segment by the previously mentioned slender branch of cuticle and a pair of ventral sclerites. The base of each of these sclerites is attached to the soft articulating cuticle that lines the vulva and is continuous with the soft cuticle lining the vagina. When using the scanning electron microscope to compare the external female genitalia in fourteen species of
As is the case for the dorsal genital segment above it, the third valvula has an overall triangular shape, but is smaller and displays a medial line that separates the sclerite into two distinct halves ( Figure 5 ). The two halves are joined only from the anterior base of the sclerite to approximately 1/3 their length, beyond which they are completely separated. The lateral and distal margins of each half forms a thick rounded edge which possesses several long fine hairs. The similarity of these hairs to tactile sensors on the insect cuticle suggests that they have a sensory function, and the manner by which they line the edge of the third valvula suggests that this structure serves as a sensory organ.
The second valvula consists of a pair of sclerites that line the lateral edges of the vulva. They are bilaterally symmetrical and elongated or lacinate in shape. They are widest at their base where they attach to the soft cuticle lining of the vulva. They also curve along their long axis at their base to form a short tube‐like structure, and they narrow posteriorly to a pointed end. The ventral edge of the second valvula forms a ridge along its margin, and this ridge fits into a groove that runs along the dorsal edge of the sclerite in the first valvula. As noted previously, the second valvula is attached at its base to the arm of cuticle that connects to the mid‐lateral region of the dorsal genital segment.
The first valvula consists of a bilaterally symmetrical pair of sclerites that are more triangular in shape than the lacinate lateral sclerites of the second valvula ( Figure 4B ). The dorsal edge of the sclerites of the first valvula forms the groove in which the ventral ridge on the sclerites of the second valvula slides. This ridge and groove mechanism allows the second valvula to extend beyond the posterior end of the first valvula while keeping these two sets of sclerites firmly attached. This intricate structural relationship between the first and second valvulae may be an adaptation to serve a physiological role as the egg is passing out of the vagina. For instance, the excretory pore of the cement gland is situated on the dorsal side of the vulva near the tubular bases of the second valvulae (see Figure 3 ), yet on exiting the body, the cement gland secretions appear as dabs of secretions on the ventral, not dorsal, side of the egg . In combination with the tubular nature of the base of the second valvula, and the ridge and groove mechanism, these valvulae may function to direct cement gland secretions onto the ventral surface of an egg as it passes through the vulva.
The contralateral sclerites of the first valvula are not directly attached to one other, but are connected to each other by the soft cuticle that lines the entrance of the vulva ( Figure 4 ). Thus, unlike the fused halves of the third valvula ( Figure 5 ), these sclerites can separate, and stretch apart passively, as would be expected when an egg courses through the vulva. They can also be pulled back into the body to close off the vulva when the muscles associated with the vagina contract to eject an egg. Inserting a previously laid egg through the vulva demonstrates how readily the paired sclerites of the third valvula separate to allow the egg to pass ( Figure 4B ).
3.3. Female reproductive organs
The structure and function of the internal organs of the adult female reproductive system have been well documented for
The spermathecae are one of the two accessory reproductive glands associated with the female reproductive system of
The cement gland, the other accessory reproductive gland in
A comparative work on Reduviidae bugs shows that these blood‐feeding insect vectors of Chagas disease can vary with respect to the presence of a cement gland and the morphology of their spermathecae . All Reduviidae examined possess spermathecae that are paired blind ended tubes attached to the side of the common oviduct. However, the shape and location of the distal ends of the spermathecae differ depending on the genus. In
3.4. Physiology of muscles associated with the vagina and valvulae
The physiology of the muscles associated with the vagina and valvulae in
The overall pattern of innervation in the abdomen of
Methylene blue also stains a network of fibres that extend over the base of the common oviduct and dorsal anterior region of the vagina . This network resembles the nerves stained with an antibody for proctolin . Application of various concentrations of proctolin to the preparation shows that increasing concentrations of proctolin have the same effect on tension generation as increases in electrical stimulation of the motor nerves . Thus, proctolin plays a significant role in regulating contractions of the vagina muscles in
3.5. Egg laying
According to the structure and function of the genitalia in
A mature chorionated egg is released from the base of the ovariole in the ovary and enters the lateral oviduct;
Peristaltic contractions of the lateral oviduct propels the egg into the common oviduct. These contractions may be spontaneous or evoked by a motor input;
The egg squeezes through the muscular vestibulum at the end of the common oviduct, and as it stretches the walls of the common oviduct, it stretches the opening of the attached spermathecae. This action allows for the release of some of the stored spermatozoa onto the egg. Release of spermatozoa may also be enhanced by motor stimulation of the spermathecae;
The egg stretches the vagina muscles and the nerve plexus attached to the vagina, and this stretching elicits a contraction of the vagina muscles causing them to shorten and pull the valvulae anteriorly, at which point, the valvulae stretch apart in response to the presence of the egg allowing the egg to start its descent out of the vagina. This step probably involves a stretch reflex which causes contractions of the vagina muscles since eggs are often seen in the lateral oviducts, but seldom lodged within the vagina ;
As the egg exits the vulva, secretions from the cement gland are delivered to the dorsal side of the egg, and the first and second valvulae relocate the cement gland secretion to the ventral side of the egg.
As the egg leaves through the vulva, inhibitory input can relax the vagina muscles allowing the valvulae to close off the vulva. This action, in conjunction with active retraction of the dorsal and ventral genital segments, squeezes the egg out of the vagina and onto the substrate. As the dorsal genital segment retracts, it may place pressure on the dorsal surface of the passing egg, and such pressure would ensure that the egg contacts the substrate. Two observations suggest this final action of the dorsal genital segment. First, a mature egg in the reproductive system shows no asymmetry but is equally rounded on all sides, whereas an egg which is laid has a distinct indentation on its dorsal surface as would be expected if pressure were placed on this location during its passage to the substrate. Second, this indentation appears to be directly related to the egg passing through the vulva, and not due to structural changes after being laid. In a SEM image of an egg passing out of the vulva, the exiting egg already shows a distinct indentation under the dorsal genital segment .
The role of the female reproductive system is also important in copulation and the ejection of male secretions after copulation, and these events will be considered following a description of the male reproductive system.
4. The male reproductive system
As in the case for the female genitalia this chapter simplifies the nomenclature by identifying the male genital segments according to their structure and function observed in the adult.
4.1. The male genitalia
The genitalia of the adult male are positioned on the underside of full‐sized abdominal segment 7, and consist of two segments which move together (see Figures 7 and 8 ). The anterior segment is smaller with no cuticle specializations, and serves to attach the larger posterior genital segment to abdominal segment 7. The skeletal muscles extending between the anterior genital segment and abdominal segment 7 move the genitalia enabling the male to extend his genitals away from his body and to turn them laterally to face the corresponding female genitalia. It is this anatomical arrangement that determines the side‐by‐side position copulating pairs assume ( Figure 1 ).
The posterior genital segment is twice the size of the anterior genital segment, and is rounded at its posterior end taking on a bulbous shape ( Figure 7 ). On this posterior‐rounded side, there are two bilaterally symmetrical arms of cuticle, the parameres, which are attached to the posterior lateral edges of the posterior genital segment (p in Figures 7 and 8 ). On their distal ends, the parameres possess fine hairs characteristic of sensory hairs associated with tactile stimulation in insects , and when not extended, they fit into a groove on the posterior dorsal edge of the posterior genital segment with their slightly flattened hook‐like ends facing one another ( Figure 7A ). During copulation, the male extends his parameres to make contact with the female genitalia, and during insemination, the curved blunt tips of the parameres wrap around the lateral flaps on the dorsal genital segment of the female. These parameres have been considered homologous to claspers in other insect species , but they do not appear to firmly latch on to the female . Preliminary results suggest that the parameres serve a sensory function aiding the male to determine the position of the female genitalia before and during copulation. Their position and sensory function suggest that they are homologous sensory organs to the third valvula in the female genitalia.
The posterior genital segment houses the aedeagus (see Figure 8 ). The aedeagus sits in a pocket lined with soft articulating cuticle and opened to the dorsal side of the posterior genital segment. During copulation, the aedeagus extends out of this pocket into the vagina. Viewed laterally, the aedeagus assumes a half‐moon shape ( Figure 9C ). The curved portion of the aedeagus contains an elaborate bag‐like structure formed from an invagination of soft cuticle with several overlying folds ( Figure 9B ). These folds allow the bag‐like structure to be extended or compressed perpendicular to the flow of secretion from the male reproductive organs. In dissections where the vital dye, methylene blue, is added to the exposed abdomen, this dye is picked up by the reproductive glands and carried in their ducts to the aedeagus where it ends up in the space between the bag‐like structure and the medial plate of the aedeagus. Methylene blue does not enter the bag‐like structure suggesting that this structure is not designed to receive secretions from the male reproductive organs .
The male secretions reach the aedeagus through the ejaculatory duct which is the fused portion of the left and right ejaculatory bulbs. This duct is anchored to a ring of cuticle in the basiphallus which serves as the supporting base for the aedeagus. A pliable delicate duct extends from this ring into the aedeagus ( Figure 9A ), and carries secretions from the ejaculatory bulb into the aedeagus when the aedeagus is extended into the vagina. Since the secretions can be deposited in the space between the bag‐like structure and the medial plate on the straight side of the aedeagus, the bag‐like structure could serve as part of a pumping mechanism that forces secretions out of the aedeagus and into the vagina during the power stroke, but prevents back flow during the recovery phase of the pumping cycle.
4.2. The male reproductive organs
The male reproductive system anterior to the ejaculatory duct is bilaterally symmetrical and each side consists of two reproductive organs—the testis and the seminal vesicle, and two types of accessory reproductive organs—the three lobes of the transparent accessory reproductive gland (tag) and the one lobe of the opaque accessory reproductive gland (oag) (see Figure 10 ). The testes are located laterally near the mid‐region of the abdomen. They are present in the penultimate larval stage (L5), and become connected to the seminal vesicle during metamorphosis. The testis consists of seven follicles folded onto each other and wrapped with a thin membrane. Two of the seven have a larger girth and a longer length than the other five, even in the L5 stage. As the testes increase in size during the adult stage, the growth is mainly attributed to the two larger follicles which increase considerably in length and girth , which has also been observed in other species of Reduviidae bugs .
Each testis is connected to the seminal vesicle by the vas deferens which extends a short distance from the testis, where the bases of the follicles are attached, to the tip of the anterior lobe of the tag. From this point, the vas deferens remains closely associated with the tag and courses along its side to the level of the lobe's base where the vas deferens connects to the seminal vesicle. Between the testis and the tip of the tag, the contents of the vas deferens tend to be transparent. From the tip of the lobe to the seminal vesicle, the contents are distinctly yellowish white and have a clump‐like appearance. The seminal vesicle is a semi‐rigid elongated sack which can increase considerably in girth as the adult matures. Its length is approximately the same length as the individual lobes of the tag ( Figure 10 ), and its duct connects to the ejaculatory bulb posterior to the duct from the accessory reproductive glands.
Of the two types of accessory reproductive glands, the larger tag consists of three large tube‐like lobes, and the smaller oag is a single elongated structure ( Figure 10 ). The tag contains a clear proteinaceous material, and the oag contains a whitish milky substance, both of which are delivered to the female during copulation. Rather than sequestering from the haemolymph molecules made from another organ or tissue, the tag may make the secretions themselves , with their activity being under endocrine control [40, 41]. They also produce a polypeptide that is secreted into the haemolymph . The tag possesses a relatively tough muscular lining that is supplied by motor axons which, when electrically stimulated, will cause each of the three lobes of the tag to constrict their girth and lengthen (personal observations). The tag secretions are viscous and pour slowly out of the lobe when it is cut. In contrast, the oag has a delicate lining, is easily damaged during dissection and its whitish secretions readily flow out of the lobe. It is widest at its anterior base, tapers towards its posterior end, and does not respond to electrical stimulation of the abdominal nerves. Early studies report that placing the contents of the oag onto an adult vagina can elicit strong twitch‐like contractions of the vestibulum suggesting that this male secretion may aid delivery of the transferred spermatozoa to the spermathecae . Because the response is described as capricious rather than consistent, this role is speculative.
Each lobe of each accessory reproductive gland empties through its own duct, and these ducts enter a tube which makes up the proximal end of the common accessory reproductive gland duct ( Figure 9D ). As these ducts enter this tube, they do not merge into a single duct at this point, but extend down the tube to become a single lumen before emptying into the ejaculatory bulb. This tube has a muscular sheath, and displays spontaneous contractions that tend to shorten the tube pulling it posteriorly towards the ejaculatory bulb.
4.3. Delivery of male secretions to the vagina
The manner by which the male secretions are delivered to the female reproductive system in insects varies between two extremes. At the one extreme, the female has two genital openings, one to the bursa copulatrix, and the other to the egg pore. The male produces a distinct spermatophore which is a proteinaceous package containing spermatozoa and this package is deposited into the bursa copulatrix. There, the spermatophore is broken open allowing the spermatozoa to migrate along the sperm duct to the spermatheca . At the other extreme, the female has a single opening to her reproductive system. The male inserts a long intromittent organ through the vagina, into an insemination duct which leads to an elaborate spermatheca. At the end of the insemination duct, the male extends his intromittent organ through a valve and pumps his secretions directly into the spermatheca. No spermatophore is needed .
The first description of sperm transfer in
Considering these more recent findings, it is likely that all Reduviidae bugs lack spermatophore sacs, and the structure thought to be a sac is part of a pumping mechanism which enables the aedeagus to fill the vagina with the male secretions. Rather than resembling those arthropods which make encapsulated spermatophores that harden before they are inserted into the bursa copulatrix of the female , the Reduviidae are more closely related to insect species which lack spermatophores and deliver the semen by using a long intromittent organ that the male inserts through an insemination duct to the spermatheca . In
As the male secretions are delivered to the female, they assume the pear‐shape of the inside of the vagina with the narrower anterior end resulting from the male secretions being pushed up against the narrow base of the common oviduct ( Figure 11 ). In a recently inseminated female, the secretions from the seminal vesicle appear as a clump of yellowish material at the base of the common oviduct whereas the rest of the vagina is filled with a slightly cloudy secretion from the accessory reproductive glands. Since the spermatozoa are positioned anteriorly, the seminal vesicle secretions are delivered first, followed by secretions of the accessory reproductive glands. With separate ducts to the ejaculatory bulb, differential motor activity from the central nervous system likely stimulates the seminal vesicles to deliver their secretions prior to transfer of the accessory reproductive gland secretions. In addition, the clump from the seminal vesicle is approximately the same size as the aedeagus suggesting that only one or two pulses from the pump in the aedeagus are needed to deliver the spermatozoa.
The remainder of the spermatophore consists of a large amount of secretion from the tag and oag. While in the body of the male, the secretions of the tag are transparent, but in the spermatophore in the female, they take on a slightly cloudy appearance ( Figure 11C ). This change likely results from a small amount of oag material mixing with a large amount of tag material which is possible due to the relationship between the ducts from the three lobes of the tag, and the single duct from the oag. All four ducts enter the distal end of the tube of the common accessory reproductive gland duct, and this tube is able to produce bursts of contractions that rhythmically constrict and shorten the tube. In addition, the lobes of the tag can contract due to motor stimulation thus forcing the material into their ducts, whereas material from the oag enters passively. This anatomical arrangement could allow the peristaltic‐like contractions of the common duct to ‘milk’ the ducts of the four lobes of accessory reproductive glands at the same time resulting in a large amount of tag material being mixed with a small amount of oag material before they are delivered to the aedeagus and the vagina. This scenario, which is supported by the anatomy and physiology, suggests that the oag secretions are affecting the tag secretions rather than eliciting contractions of the vestibulum. Davey  postulated that the secretions from the cells lining the ejaculatory bulb mix with the tag secretions to lower the pH, causing the tag secretions to harden. However, the oag secretions may also serve in hardening the tag secretions. Determining the relationship between the secretions from the tag and oag promises to be a fruitful area of study.
4.4. Facilitating copulation
If the sensory hairs on the ventral lateral region of the abdomen are gently stroked with a probe, the heartbeat is inhibited . Such a reflex could be part of a general thigmotactic response in which the insect becomes less responsive to external stimuli when it wedges itself into a confined space (see p. 313 in Ref. ). This response could be elicited as these sensory hairs touch the surface of the enclosed area, and the stoppage of the heart beat may be part of the general calming of the whole body. The ventral region of the abdomen linked to this tactile inhibition of the heartbeat is the same region where the male places his abdomen during copulation, which, in turn, could generate a thigmotactic response to help calm the female.
This sensory thigmotactic response could be enhanced chemically by rhodtestolin, a cardio‐inhibitor first discovered in testes extracts of
Our knowledge of the details of sexual reproduction in
This knowledge gained by detailing the mechanics of copulation and egg‐laying in this well‐studied insect sets the groundwork from which further investigation of this important physiological process in this bug can be carried out. Along with the completion of the