Relationships between altitude, triatomine (Triatoma dimidiata) immune response and virulence of Trypanosoma cruzi, the causal agent of Chagas' disease.

Little is known about how the virulence of a human pathogen varies in the environment it shares with its vector. This study focused on whether the virulence of Trypanosoma cruzi (Trypanosomatida: Trypanosomatidae), the causal agent of Chagas' disease, is related to altitude. Accordingly, Triatoma dimidiata (Hemiptera: Reduviidae) specimens were collected at three different altitudes (300, 700 and 1400 m a.s.l.) in Chiapas, Mexico. The parasite was then isolated to infect uninfected T. dimidiata from the same altitudes, as well as female CD-1 mice. The response variables were phenoloxidase (PO) activity, a key insect immune response, parasitaemia in mice, and amastigote numbers in the heart, oesophagus, gastrocnemius and brain of the rodents. The highest levels of PO activity, parasitaemia and amastigotes were found for Tryp. cruzi isolates sourced from 700 m a.s.l., particularly in the mouse brain. A polymerase chain reaction-based analysis indicated that all Tryp. cruzi isolates belonged to a Tryp. cruzi I lineage. Thus, Tryp. cruzi from 700 m a.s.l. may be more dangerous than sources at other altitudes. At this altitude, T. dimidiata is more common, apparently because the conditions are more beneficial to its development. Control strategies should focus activity at altitudes around 700 m a.s.l., at least in relation to the region of the present study sites.

Origin, evolution and function of the hemipteran perimicrovillar membrane with emphasis on Reduviidae that transmit Chagas disease.

The peritrophic matrix is a chitin-protein structure that envelops the food bolus in the midgut of the majority of insects, but is absent in some groups which have, instead, an unusual extra-cellular lipoprotein membrane named the perimicrovillar membrane. The presence of the perimicrovillar membrane (PMM) allows these insects to exploit restricted ecological niches during all life stages. It is found only in some members of the superorder Paraneoptera and many of these species are of medical and economic importance. In this review we present an overview of the midgut and the digestive system of insects with an emphasis on the order Paraneoptera and differences found across phylogenetic groups. We discuss the importance of the PMM in Hemiptera and the apparent conservation of this structure among hemipteran groups, suggesting that the basic mechanism of PMM production is the same for different hemipteran species. We propose that the PMM is intimately involved in the interaction with parasites and as such should be a target for biological and chemical control of hemipteran insects of economic and medical importance.

To be or not to be? Mating success and survival trade-offs when switching between alternative reproductive tactics.

Hormones underlie the decision of assuming a territorial or a nonterritorial role, with territorial individuals usually having higher hormonal levels than nonterritorial individuals. As a territorial status is linked to higher mating opportunities, it is unclear why animals do not keep high hormonal levels and one explanation is that this would imply survival costs. We have tested this using males of the territorial damselfly Argia emma in the field. We increased juvenile hormone (JH) levels using methoprene in both territorial and nonterritorial males and predicted that: (i) males will keep (the case of territorial males) or become (the case of nonterritorial males) territorial after hormonal increase, and (ii) there will be an increase in mating success for nonterritorial males only and an impaired survival for both male tactics. Hormonally treated males remained or became territorial but had their survival impaired compared with control groups. Also, hormonally treated, ex-nonterritorial males increased their mating success compared with the other control, nonterritorial males. The reduced survival can be explained proximally by the energy devoted either to the enhanced aggression showed during territory defence or immune function (as detected previously in damselflies). Although nonterritorial males may increase their mating success by switching to a territorial tactic, they are possibly unable to do it naturally as JH is dietary dependent and usually nonterritorial animals are in poorer condition than territorial animals.

Immune defence mechanisms of triatomines against bacteria, viruses, fungi and parasites.

Triatomines are vectors that transmit the protozoan haemoflagellate Trypanosoma cruzi, the causative agent of Chagas disease. The aim of the current review is to provide a synthesis of the immune mechanisms of triatomines against bacteria, viruses, fungi and parasites to provide clues for areas of further research including biological control. Regarding bacteria, the triatomine immune response includes antimicrobial peptides (AMPs) such as defensins, lysozymes, attacins and cecropins, whose sites of synthesis are principally the fat body and haemocytes. These peptides are used against pathogenic bacteria (especially during ecdysis and feeding), and also attack symbiotic bacteria. In relation to viruses, Triatoma virus is the only one known to attack and kill triatomines. Although the immune response to this virus is unknown, we hypothesize that haemocytes, phenoloxidase (PO) and nitric oxide (NO) could be activated. Different fungal species have been described in a few triatomines and some immune components against these pathogens are PO and proPO. In relation to parasites, triatomines respond with AMPs, including PO, NO and lectin. In the case of T. cruzi this may be effective, but Trypanosoma rangeli seems to evade and suppress PO response. Although it is clear that three parasite-killing processes are used by triatomines - phagocytosis, nodule formation and encapsulation - the precise immune mechanisms of triatomines against invading agents, including trypanosomes, are as yet unknown. The signalling processes used in triatomine immune response are IMD, Toll and Jak-STAT. Based on the information compiled, we propose some lines of research that include strategic approaches of biological control.

Genetic divergence predicts reproductive isolation in damselflies.

Reproductive isolation is the defining characteristic of a biological species, and a common, but often untested prediction is a positive correlation between reproductive isolation and genetic divergence. Here, we test for this correlation in odonates, an order characterized by strong sexual selection. First, we measure reproductive isolation and genetic divergence in eight damselfly genera (30 species pairs) and test for a positive correlation. Second, we estimate the genetic threshold preventing hybrid formation and empirically test this threshold using wild populations of species within the Ischnura genus. Our results indicate a positive and strong correlation between reproductive isolation and genetic distance using both mitochondrial and nuclear genes cytochrome oxidase II (COII: r = 0.781 and 18S-28S: r = 0.658). Hybridization thresholds range from -0.43 to 1.78% for COII and -0.052-0.71% for 18S-28S, and both F1 -hybrids and backcrosses were detected in wild populations of two pairs of Ischnura species with overlapping thresholds. Our study suggests that threshold values are suitable to identify species prone to hybridization and that positive isolation-divergence relationships are taxonomically widespread.

Effect of juvenile hormone on senescence in males with terminal investment.

Senescence, a decline in survival and reproductive prospects with age, is controlled by hormones. In insects, juvenile hormone (JH) is involved in senescence with captive individuals, but its effect under natural conditions is unknown. We have addressed this gap by increasing JH levels in young and old wild males of the damselfly Hetaerina americana. We assessed survival in males that were treated with a JH analogue (methoprene), which is known to promote sexual activity, and an immune challenge, which is known to promote terminal investment in reproduction in the studied species. We replicated the same procedure in captivity (to control for environmental variation), where males were deprived of any activity or food. We expected old males to show the lowest survival after being treated with JH and immune-challenged, because the effect of terminal investment on senescence would be exacerbated by JH. However, this should be the case for wild animals, but not for captive animals, as the effects of JH and immune challenge should lead to an increase in high energetic-demanding activities only occurring in the wild. Old animals died sooner compared with young animals in both the wild and captivity, confirming that males are subject to senescence. In wild but not captive animals, JH decreased survival in young males and increased it in old males, confirming that JH is sensitive to the environment when shaping animal senescence. Immune challenge had no effect on survival, suggesting no effect of terminal investment on senescence. Additionally, contrary to the expected effects of terminal investment, with an immune challenge, recapture rates increased in young males and decreased in old males. Our results show that male senescence in the wild is mediated by JH and that terminal investment does not cause senescence. One explanation is that animals undergoing senescence and terminal investment modify their feeding behaviour to compensate for their physiological state.

Maintenance of polymorphic females: do parasites play a role?

The role of parasites in explaining maintenance of polymorphism is an unexplored research avenue. In odonates, female-limited color polymorphism (one female morph mimicking the conspecific male and one or more gynochromatic morphs) is widespread. Here we investigated whether parasitism contributes to color polymorphism maintenance by studying six species of female dimorphic damselflies using large databases of field-collected animals. We predicted that androchrome females (male mimics) would be more intensively parasitized than gynochrome females which is, according to previous studies, counterbalanced by the advantages of the former when evading male harassment compared to gynochrome females. Here we show that in Ischnura denticollis and Enallagma novahispaniae, androchrome females suffer from a higher degree of parasitism than gynochromatic females, and contrary to prediction, than males. Thus, our study has detected a correlation between color polymorphism and parasitic burden in odonates. This leads us to hypothesize that natural selection, via parasite pressure, can explain in part how androchrome and gynochrome female color morphs can be maintained. Both morphs may cope with parasites in a different way: given that androchrome females are more heavily parasitized, they may pay a higher fecundity costs, in comparison to gynochrome females.

Current immunity markers in insect ecological immunology: assumed trade-offs and methodological issues.

The field of ecological immunology currently relies on using a number of immune effectors or markers. These markers are usually used to infer ecological trade-offs (via conflicts in resource allocation), though physiological nature of these markers remains elusive. Here, we review markers frequently used in insect evolutionary ecology research: cuticle darkening, haemocyte density, nodule/capsule formation, phagocytosis and encapsulation/melanization via use of nylon filaments and beads, phenoloxidase activity, nitric oxide production, lysozyme and antimicrobial peptide production. We also provide physiologically based information that may shed light on the probable trade-offs inferred when these markers are used. In addition, we provide a number of methodological suggestions to improve immune marker assessment.

Allometry in damselfly ornamental and genital traits: solving some pitfalls of allometry and sexual selection.

Static allometry of sexually selected traits has been the subject of intense research recently. However, some pitfalls for this kind of research are: (a) the functions of sexual traits are largely unknown; (b) more than one body size indicator must be measured; and, (c) allometry must be examined under different environmental circumstances to see whether allometric values change. Using Hetaerina americana damselflies, we investigated the type of allometry exhibited by a wing red spot and aedeagal width. These traits are positively selected during pre-copulatory male-male contests and post-copulatory female stimulation, respectively. As body size indicators, we used wing length and head width. It has been documented that expression of both sexual traits varies throughout the year. Thus, allometry was examined in different times of the year. We also investigated the allometry of aedeagal width and vaginal width at the zone where female stimulation takes place. We found no clear pattern of any allometric relationship for male and female traits and for both body size indicators at all times sampled. Our results contrast with patterns of negative allometry exhibited by genital traits in other animals.

Do individuals in better condition survive for longer? Field survival estimates according to male alternative reproductive tactics and sex.

There is a gap in terms of the supposed survival differences recorded in the field according to individual condition. This is partly due to our inability to assess survival in the wild. Here we applied modern statistical techniques to field-gathered data in two damselfly species whose males practice alternative reproductive tactics (ARTs) and whose indicators of condition in both sexes are known. In Paraphlebia zoe, there are two ART: a larger black-winged (BW) male which defends mating territories and a smaller hyaline-winged (HW) male that usually acts as a satellite. In this species, condition in both morphs is correlated with body size. In Calopteryx haemorrhoidalis, males follow tactics according to their condition with males in better condition practicing a territorial ART. In addition, in this species, condition correlates positively with wing pigmentation in both sexes. Our prediction for both species was that males practicing the territorial tactic will survive less longer than males using a nonterritorial tactic, and larger or more pigmented animals will survive for longer. In P. zoe, BW males survived less than females but did not differ from HW males, and not necessarily larger individuals survived for longer. In fact, size affected survival but only when group identity was analysed, showing a positive relationship in females and a slightly negative relationship in both male morphs. For C. haemorrhoidalis, survival was larger for more pigmented males and females, but size was not a good survival predictor. Our results partially confirm assumptions based on the maintenance of ARTs. Our results also indicate that female pigmentation, correlates with a fitness component - survival - as proposed by recent sexual selection ideas applied to females.

Genital morphology and copulatory behavior in triatomine bugs (Reduviidae: Triatominae).

Triatomines (Heteroptera: Reduviidae) include around 139 species, widely known as vectors of Chagas disease. Our aim is to review the existing knowledge of the genital morphology and sexual behavior and provide some functional analysis of these traits in triatomines. A complex set of traits comprise genitalia and these are highly variable among species. The components of the phallus and seminal products (secreted by action of testes and two accessory glands) interact to allow successful sperm transfer to the female spermathecae (usually a pair of blind tubes that emerge from the common oviduct). Seminal products may inhibit female physiology and extend mating duration. Mating behavior in triatomines is best characterized as scramble competition. We suggest that males may evaluate female condition prior to copulation, given that female fitness is largely affected by food (blood) source. Although rearing several triatomine species may be difficult and discourage from undertaking studies on this group, any further investigation on sexual behavior and mating interactions may provide data for applicative studies including Chagas disease vectors control.

Sexual selection, sexual size dimorphism and Rensch's rule in Odonata.

Odonata (dragonflies and damselflies) exhibit a range of sexual size dimorphism (SSD) that includes species with male-biased (males > females) or female-biased SSD (males < females) and species exhibiting nonterritorial or territorial mating strategies. Here, we use phylogenetic comparative analyses to investigate the influence of sexual selection on SSD in both suborders: dragonflies (Anisoptera) and damselflies (Zygoptera). First, we show that damselflies have male-biased SSD, and exhibit an allometric relationship between body size and SSD, that is consistent with Rensch's rule. Second, SSD of dragonflies is not different from unit, and this suborder does not exhibit Rensch's rule. Third, we test the influence of sexual selection on SSD using proxy variables of territorial mating strategy and male agility. Using generalized least squares to account for phylogenetic relationships between species, we show that male-biased SSD increases with territoriality in damselflies, but not in dragonflies. Finally, we show that nonagile territorial odonates exhibit male-biased SSD, whereas male agility is not related to SSD in nonterritorial odonates. These results suggest that sexual selection acting on male sizes influences SSD in Odonata. Taken together, our results, along with avian studies (bustards and shorebirds), suggest that male agility influences SSD, although this influence is modulated by territorial mating strategy and thus the likely advantage of being large. Other evolutionary processes, such as fecundity selection and viability selection, however, need further investigation.

The expression of a sexually selected trait correlates with different immune defense components and survival in males of the American rubyspot.

Recent studies have suggested that courtship trait expression indicates immune strength. However, most studies have measured only one immune parameter, have not assessed individual differences in immune ability according to time and have not controlled for ecological differences among individuals after an immune challenge. In this work, we tested this hypothesis and controlled for these factors using males of the American rubyspot damselfly which bear a wing red spot whose size is evolutionarily maintained via male-male territorial competition. Our general hypothesis was that territorial, large-spotted males, had a better immune ability compared to nonterritorial, small-spotted males. We expected that the following variables were greater in territorial males compared to nonterritorial males: spot size, phenoloxidase (PO) and hydrolytic enzymatic (HE) activity in males challenged and nonchallenged with a nylon implant, PO and HE activity rate; PO activity after a Serratia marcescens challenge, and survival after a nylon challenge controlling for activity and feeding differences. We found that territorial males showed larger spot areas, greater PO and HE activity (independently of whether they were challenged or not), a higher rate of PO and HE activity (but only expressed at 8h), greater PO production after the bacterial challenge, and a higher survival after the challenge. These results corroborate that males with more pronounced sexual traits have a superior immune function.

Sexual comparisons in immune ability, survival and parasite intensity in two damselfly species.

Recent evolutionary studies have suggested that females have a more robust immune system than males. Using two damselfly species (Hetaerina americana and Argia tezpi), we tested if females produced higher immune responses (as phenoloxidase and hydrolytic enzymes), had a higher survival (using a nylon implant inserted in the abdomen and measuring survival after 24h) and fewer parasites (gregarines and water mites) than males. We also tested whether immune differences should emerge in different body areas (thorax vs. abdomen) within each sex with the prediction that only females will differ with the abdomen having a higher immune response than their thorax since the former area, for ecological and physiological reasons, may be a target zone for increased immune investment. Animals were adults of approximately the same age. In both species, females were more immunocompetent than males, but only in H. americana females were immune responses greater in the abdomen than in the thorax. However, there were no differences in survival and parasite intensity or the probability of being parasitised between the sexes in either of the two species. Thus, this study lends partial support to the principle that females are better at defending than males despite the null difference in parasitism and survival.

Immune Priming, Fat Reserves, Muscle Mass and Body Weight of the House Cricket is Affected by Diet Composition.

Some insect species are capable of producing an enhanced immune response after a first pathogenic encounter, a process called immune priming. However, whether and how such ability is driven by particular diet components (protein/carbohydrate) have not been explored. Such questions are sound given that, in general, immune response is dietary dependent. We have used adults of the house cricket Acheta domesticus L. (Orthoptera: Gryllidae) and exposed them to the bacteria Serratia marcescens. We first addressed whether survival rate after priming and nonpriming treatments is dietary dependent based on access/no access to proteins and carbohydrates. Second, we investigated how these dietary components affected fat reserves, muscle mass, and body weight, three key traits in insect fitness. Thus, we exposed adult house crickets to either a protein or a carbohydrate diet and measured the three traits. After being provided with protein, primed animals survived longer compared to the other diet treatments. Interestingly, this effect was also sex dependent with primed males having a higher survival than primed females when protein was supplemented. For the second experiment, protein-fed animals had more fat, muscle mass, and body weight than carbohydrate-fed animals. Although we are not aware of the immune component underlying immune priming, our results suggest that its energetic demand for its functioning and/or consequent survival requires a higher demand of protein with respect to carbohydrate. Thus, protein shortage can impair key survival-related traits related to immune and energetic condition. Further studies varying nutrient ratios should verify our results.

Temporal Variation in Immune Components of the White Grub Phyllophaga polyphylla (Bates) (Coleoptera: Melolonthidae).

Ecological immunology assumes that the host immune efficiency is correlated with its survival after pathogen challenge. To test this hypothesis, we challenged Phyllophaga polyphylla (Bates) larvae with the naturally occurring fungus Metarhizium pingshaense on two consecutive years (2011 and 2012). In each year, we injected the blastospores of M. pingshaense and then used levels of prophenoloxidase (proPO), phenoloxidase (PO) and total haemolymph serum protein as indicators of immune efficiency. Larvae were injected with (1) phosphate buffered saline (PBS) + Tween and viable blastospores of M. pingshaense, (2) PBS + Tween and non-viable blastospores of M. pingshaense, (3) PBS + Tween, or (4) non-manipulated. Overall, levels of PO, proPO and total haemolymph serum protein in larvae after 12 h were similar amongst treatments within each year of collection. However, larvae collected in 2011 showed higher PO and proPO activity but lower total haemolymph serum protein compared with larvae collected in 2012. A survival study injecting viable blastospores showed that larvae collected in both years died within 48 h; however, when non-viable blastospores were injected, which were still toxic to larvae, mortality was greater in larvae collected in 2011 compared with larvae collected in 2012. Altogether, these results indicate that PO, proPO and total haemolymph serum protein do not predict immune strength of P. polyphylla against blastospores of M. pingshaense, but higher values of PO and proPO were correlated with higher survival rates against non-infective but toxic agents. The possible role of some abiotic factors over the differences observed for immune components of P. polyphylla in different years of collection is discussed.

Postmating sexual selection: allopatric evolution of sperm competition mechanisms and genital morphology in calopterygid damselflies (Insecta: Odonata).

Postmating sexual selection theory predicts that in allopatry reproductive traits diverge rapidly and that the resulting differentiation in these traits may lead to restrictions to gene flow between populations and, eventually, reproductive isolation. In this paper we explore the potential for this premise in a group of damselflies of the family Calopterygidae, in which postmating sexual mechanisms are especially well understood. Particularly, we tested if in allopatric populations the sperm competition mechanisms and genitalic traits involved in these mechanisms have indeed diverged as sexual selection theory predicts. We did so in two different steps. First, we compared the sperm competition mechanisms of two allopatric populations of Calopteryx haemorrhoidalis (one Italian population studied here and one Spanish population previously studied). Our results indicate that in both populations males are able to displace spermathecal sperm, but the mechanism used for sperm removal between both populations is strikingly different. In the Spanish population males seem to empty the spermathecae by stimulating females, whereas in the Italian population males physically remove sperm from the spermathecae. Both populations also exhibit differences in genital morphometry that explain the use of different mechanisms: the male lateral processes are narrower than the spermathecal ducts in the Italian population, which is the reverse in the Spanish population. The estimated degree of phenotypic differentiation between these populations based on the genitalic traits involved in sperm removal was much greater than the differentiation based on a set of other seven morphological variables, suggesting that strong directional postmating sexual selection is indeed the main evolutionary force behind the reproductive differentiation between the studied populations. In a second step, we examined if a similar pattern in genital morphometry emerge in allopatric populations of this and other three species of the same family (Calopteryx splendens, C. virgo and Hetaerina cruentata). Our results suggest that there is geographic variation in the sperm competition mechanisms in all four studied species. Furthermore, genitalic morphology was significantly divergent between populations within species even when different populations were using the same copulatory mechanism. These results can be explained by probable local coadaptation processes that have given rise to an ability or inability to reach and displace spermathecal sperm in different populations. This set of results provides the first direct evidence of intraspecific evolution of genitalic traits shaped by postmating sexual selection.

Sensory trap as the mechanism of sexual selection in a damselfly genitalic trait (Insecta: Calopterygidae).

During copulation, males of some calopterygid damselfly species displace the sperm stored in the spermatheca: the male genital appendages enter into the spermathecal ducts and physically remove sperm. In Calopteryx haemorrhoidalis, the genital appendages are too wide to penetrate the spermathecae, but males use a different mechanism in which the aedeagus stimulates the vaginal sensilla that control spermathecal sperm release. Since these sensilla are used during egg fertilization and oviposition, it was hypothesized that this function evolved before the male stimulatory ability. I investigated this using Hetaerina cruentata, a species whose position in the Calopterygidae phylogeny is more basal than Calopteryx. Given this position and having determined that males of this species are not able to displace sperm of their conspecific females during copulation, it was expected that H. cruentata females would eject sperm when stimulated with the aedeagi of C. haemorrhoidalis but not when stimulated with the aedeagi of their conspecifics. This prediction was confirmed. In order to investigate the widespread nature of this result, some other Calopteryx species-Calopteryx xanthostoma and Calopteryx virgo-were investigated. The results were similar to those of H. cruentata: conspecific males were unable to stimulate their females, but females ejected sperm when stimulated with C. haemorrhoidalis aedeagi. Morphometric analysis suggests that the mechanistic explanation for the stimulatory ability of C. haemorrhoidalis genitalia is that the aedeagal region that makes contact with the vaginal sensilla is wider in C. haemorrhoidalis than in the other species. These results suggest that the sensory "bias" shown and shared by H. cruentata, Calopteryx splendens, C. virgo, and C. haemorrhoidalis females represents an ancestral condition and that the male stimulatory ability is absent in the evolutionary history of the clade. These pieces of evidence as well as another one presented elsewhere, which indicates that C. haemorrhoidalis males vary in their stimulatory ability, constitute the three criteria for a case of sexual selection via exploitation of a female sensory bias. These results also provide support to the sensory trap hypothesis that indicates that the female bias-in this case, egg fertilization and oviposition-evolved in a context different from sexual selection. Considering that the male genital appendages responsible for physically removing spermathecal sperm in other calopterygids are present in C. haemorrhoidalis, I suggest that males were once able to displace spermathecal sperm physically. Such ability may have been later impeded by a reduction in size of the spermathecal ducts. Possibly, one of the latest events in this sequence is the male's stimulatory ability. This hypothetical series of events suggests a coevolutionary scenario in which the central actor is the sperm stored in the spermathecae.

No Detectable Trade-Offs Among Immune Function, Fecundity, and Survival via a Juvenile Hormone Analog in the House Cricket.

Hormones are key regulators of resource allocation among functions and thus play an important role in resource-based trade-offs. The juvenile hormone (JH) is an insect hormone that mediates resource allocation between immunity and life history components. Here, we have tested whether this is the case using the house cricket. We investigated whether increased levels of JH (using methoprene, a JH analog) enable an enhanced survival and fecundity (via egg number) at the cost of reduced hemocyte number (a trait that is associated with immune response in insects) in the house cricket, Acheta domesticus L. We had three groups of adult crickets of both sexes: experimental (methoprene and acetone), positive control (methoprene), and negative control (no manipulation). Prior to and after experimental treatments, we counted the number of hemocytes (for the case of both sexes) and recorded the number of eggs laid and survival of females after the manipulation. There was no difference in hemocyte number, egg number, and survival. These results do not support a JH-mediated trade-off among immune ability, survival, and fecundity. We provide arguments to explain the lack of JH-mediated trade-offs in the house cricket.

Possible coevolution of male and female genital form and function in a calopterygid damselfly.

In this paper some evolutionary changes of genitalia in the damselfly Calopteryx haemorrhoidalis are investigated by determining their current and past function. Calopteryx haemorrhoidalis males stimulate females by aedeagal frictioning on a set of vaginal sensilla. The aedeagus is considerably variable and positively correlates with volumes of ejected sperm from the spermatheca. Interestingly, females show a significantly reduced sensillum number compared with other family members. Here I explore whether there existed directional selection for aedeagal width at its evolutionary onset; and whether the sensillum reduction evolved to make sperm ejection less effective. Using C. haemorrhoidalis aedeagi in females whose species retained the ancestral conditions (no stimulatory ability and large sensillum numbers), Hetaerina cruentata and C. xanthostoma, my results corroborated these assumptions: variation in aedeagal width inversely correlated with sperm ejection rate while sperm ejection was higher in species with high sensillum numbers. A suggested coevolutionary interpretation of these results in C. haemorrhoidalis is that aedeagal width was favoured which was followed by a sensillum reduction.