Secondary contact zone and genetic introgression in closely related haplodiploid social spider mites.

How frequently hybridisation and gene flow occur in the contact zones of diverging taxa is important for understanding the speciation process. Stigmaeopsis sabelisi and Stigmaeopsis miscanthi high-aggression form (hereafter, S. miscanthi HG) are haplodiploid, social spider mites that infest the Chinese silver grass, Miscanthus sinensis. These two species are closely related and parapatrically distributed in Japan. In mountainous areas, S. sabelisi and S. miscanthi HG are often found in the highlands and lowlands, respectively, suggesting that they are in contact at intermediate altitudes. It is estimated that they diverged from their common ancestors distributed in subtropical regions (south of Japan) during the last glacial period, expanded their distribution into the Japanese Archipelago, and came to have such a parapatric distribution (secondary contact). As their reproductive isolation is strong but incomplete, hybridisation and genetic introgression are expected at their distributional boundaries. In this study, we investigated their spatial distribution patterns along the elevation on Mt. Amagi using male morphological differences, and investigated their hybridisation status using single-nucleotide polymorphisms by MIG-seq. We found their contact zone at altitudes of 150-430 m, suggesting that their contact zone is prevalent in the parapatric area, which is in line with a previous study. Interspecific mating was predicted based on the sex ratio in the contact zone. No obvious hybrids were found, but genetic introgression was detected although it was extremely low.

Sperm-depleted males of the two-spotted spider mite can replenish sperm in a few hours.

In many animals, males increase their reproductive success by mating with as many females as possible. The number of females a male can fertilize is often limited by male competition for access to females, sperm competition, and the cost of sperm production. Especially, recent studies have shown that sperm production is more costly than previously expected. In the two-spotted spider mite, Tetranychus urticae Koch, the number of females a male can inseminate is limited mainly by male competition for access to females. However, in the absence of rivals, males mate with so many females that they can become sperm-depleted. Mating without sperm transfer does not produce any offspring, although it takes time and energy. Therefore, a question arises as to why males continue to mate even after sperm depletion. In this study, we hypothesized that males continue to mate because sperm is replenished after a short period. To test the hypothesis, we investigated how long it takes for sperm replenishment after sperm depletion. We found that in 3 h, sperm can be replenished enough to inseminate a few females. As 3 h is sufficiently short not to lose the next mating opportunity, the results support the hypothesis. However, copulation duration in the sperm-replenished males was significantly longer than in the sperm-depleted males but shorter than in males before sperm depletion. To explain the differences, further research would be necessary. In addition, anatomical physiology study in males is also required to confirm that sperm is indeed depleted and replenished.

Males mate with females even after sperm depletion in the two-spotted spider mite.

Generally, males increase their reproductive success by mating with as many females as possible, whereas females increase their reproductive success by choosing males who provide more direct and indirect benefits. The difference in reproductive strategy between the sexes creates intense competition among males for access to females, therefore males spend much energy and time for competition with rival males for their reproduction. However, if they do not need to engage themselves into male competition and females are in no short supply, how many females can a male mate with and fertilize? We address this question in the two-spotted spider mite, Tetranychus urticae Koch. In this study, we investigated how many females a young, virgin male mated in 3 h, and checked whether the mated females were fertilized. We found that on average males mated with 12-13 females (range: 5-25). As latency to next mating did not change with the number of matings, the males are predicted to engage in even more matings if the mating trial were continued beyond 3 h. Copulation durations decreased with the number of matings and typically after 11 copulations with females any further copulations did not lead to fertilization, suggesting that males continued to mate with females even after sperm depletion. We discuss why spider mite males continue to display mating and copulation behaviour even after their sperm is depleted.

Reproductive interference and sensitivity to female pheromones in males and females of two herbivorous mite species.

Competitive interaction between sister species can be affected by reproductive interference (RI) depending on the ability of males to discriminate conspecific from heterospecific mates. We study such interactions in Tetranychus evansi and T. urticae. These spider mites co-occur on solanaceous plants in Southern Europe, and cause important yield losses in tomato crops. Previous studies using Spanish populations found that T. evansi outcompetes T. urticae, and that this is due to unidirectional RI of T. evansi males with T. urticae females. The unidirectional RI is attributed to differences in male mate preference for conspecific females between the two species. Also, differences in the propensity of interspecific web sharing in females plays a role. To investigate proximate mechanisms of this RI, here we study the role of female pheromones on male mate preference and female web sharing. We extracted pheromones from females of the two species, and investigated if males and females were arrested by the pheromone extractions in various concentrations. We observed that T. urticae males were more sensitive to the pheromone extractions and able to discriminate conspecific from heterospecific ones. Tetranychus evansi males, on the other hand, were less sensitive. Females from both species were arrested by conspecific pheromone extraction in lower concentrations. In conclusion, heterospecific mating by T. evansi males, which results in RI, can be explained by their lack of discrimination between female pheromones of the two species. Differences in the propensity of interspecific web sharing in females might not be explained by the pheromones that we investigated.

Patterns of reproductive isolation in a haplodiploid - strong post-mating, prezygotic barriers among three forms of a social spider mite.

In speciation research, much attention is paid to the evolution of reproductive barriers, preventing diverging groups from hybridizing back into one gene pool. The prevalent view is that reproductive barriers evolve gradually as a by-product of genetic changes accumulated by natural selection and genetic drift in groups that are segregated spatially and/or temporally. Reproductive barriers, however, can also be reinforced by natural selection against maladaptive hybridization. These mutually compatible theories are both empirically supported by studies, analysing relationships between intensity of reproductive isolation and genetic distance in sympatric taxa and allopatric taxa. Here, we present the - to our knowledge - first comparative study in a haplodiploid organism, the social spider mite Stigmaeopsis miscanthi, by measuring premating and post-mating, pre- and post-zygotic components of reproductive isolation, using three recently diverged forms of the mite that partly overlap in home range. We carried out cross-experiments and measured genetic distances (mitochondrial DNA and nuclear DNA) among parapatric and allopatric populations of the three forms. Our results show that the three forms are reproductively isolated, despite the absence of premating barriers, and that the post-mating, prezygotic component contributes most to reproductive isolation. As expected, the strength of post-mating reproductive barriers positively correlated with genetic distance. We did not find a clear pattern of prezygotic barriers evolving faster in parapatry than in allopatry, although one form did show a trend in line with the ecological and behavioural relationships between the forms. Our study advocates the versatility of haplodiploid animals for investigating the evolution of reproductive barriers.

Variation in nesting behavior of eight species of spider mites, Stigmaeopsis having sociality.

Nesting behavior is considered to be an important element of social living in animals. The spider mites belonging to the genus Stigmaeopsis spend their lives within nests produced from silk threads. Several of these species show cooperative sociality, while the others are subsocial. In order to identify the origins of this social behavior, comparisons of nest sizes, nesting behaviors (making nests continuously or separately), and their associated traits (fecal deposition patterns) were made for eight cogeneric Stigmaeopsis species showing various levels of social development. All of these species inhabit bamboo plants (Poaceae). We initially addressed the proximate factor of nest size variation. The variation in nest size of the eight species corresponded well with the variation in dorsal seta sc1 length, suggesting that nest size variation among species may have a genetic basis. The time spent within a nest (nest duration) increased with nest size on the respective host plants. Nest arrangement patterns varied among species showing different sized nests: Large nest builders continuously extended their nests, while middle and small nest-building species built new separate nests, which resulted in different social interaction times among species, and is thought to be closely related to social development. Fecal deposition behaviors also varied among Stigmaeopsis species, suggesting diversity in anti-predatory adaptations. Finally, we discuss how the variation in sociality observed within this genus is likely the result of nest size variation that initially evolved as anti-predator strategies.

Clinical Implications of Plasma N-acetyl-seryl-aspartyl-lysyl-proline Level in Stable Kidney Transplant Recipients.

BACKGROUND: N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is partially eliminated in urine, accumulation of AcSDKP due to chronic renal failure may cause anemia. However, the status of plasma AcSDKP level in stable kidney transplant recipients is unknown although some recipients develop anemia after kidney transplantation. In this study, we investigated the relationship between plasma AcSDKP-like immunoreactive substance (IS) level and clinical characteristics associated with renal anemia in stable kidney transplant recipients. METHODS: Forty Japanese kidney transplant recipients who underwent transplantation more than 90 days prior to the study were included. Morning blood samples were collected and plasma AcSDKP-IS levels were measured using an enzyme immunoassay. RESULTS: A significant correlation was observed between plasma AcSDKP-IS level and creatinine clearance. On the other hand, no significant correlation was observed between plasma AcSDKP-IS level and prolyl oligopeptidase activity, angiotensin II, or erythropoietin level. A significant difference in plasma AcSDKP-IS level was observed between recipients with no renal anemia and those with renal anemia. CONCLUSIONS: These results suggest that plasma AcSDKP level may depend largely on renal function and suggest a possibility that accumulation of AcSDKP may be partially involved in the pathogenesis of renal anemia in stable kidney transplant recipients.

Stalk cell differentiation without polyketides in the cellular slime mold.

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells-the major part of the prestalk cells-are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.

The role of web sharing, species recognition and host-plant defence in interspecific competition between two herbivorous mite species.

When competing with indigenous species, invasive species face a problem, because they typically start with a few colonizers. Evidently, some species succeeded, begging an answer to the question how they invade. Here, we investigate how the invasive spider mite Tetranychus evansi interacts with the indigenous species T. urticae when sharing the solanaceous host plant tomato: do they choose to live together or to avoid each other's colonies? Both species spin protective, silken webs on the leaf surfaces, under which they live in groups of con- and possibly heterospecifics. In Spain, T. evansi invaded the non-crop field where native Tetranychus species including T. urticae dominated. Moreover, T. evansi outcompetes T. urticae when released together on a tomato plant. However, molecular plant studies suggest that T. urticae benefits from the local down-regulation of tomato plant defences by T. evansi, whereas T. evansi suffers from the induction of these defences by T. urticae. Therefore, we hypothesize that T. evansi avoids leaves infested with T. urticae whereas T. urticae prefers leaves infested by T. evansi. Using wild-type tomato and a mutant lacking jasmonate-mediated anti-herbivore defences, we tested the hypothesis and found that T. evansi avoided sharing webs with T. urticae in favour of a web with conspecifics, whereas T. urticae more frequently chose to share webs with T. evansi than with conspecifics. Also, T. evansi shows higher aggregation on a tomato plant than T. urticae, irrespective of whether the mites occur on the plant together or not.

Why do males choose heterospecific females in the red spider mite?

In some species, males readily show courtship behaviour towards heterospecific females and even prefer them to females of their own species. This behaviour is generally explained by indiscriminate mating to acquire more mates, but may partly be explained by male mate preference mechanisms that have developed to choose among conspecific females, as male preference for larger females causes mating with larger heterospecific females. Recently, we found that males of the red spider mite, Tetranychus evansi collected from Spain (invasive population), prefer to mate with females of the two-spotted spider mite, T. urticae rather than with conspecific females. In spider mites, mate preference for non-kin individuals has been observed. Here, we investigated if T. evansi males collected from the area of its origin (Brazil) also show preference for heterospecific females. Secondly, we investigated if mate preference of T. evansi males for heterospecific females is affected by their relatedness to conspecific females which are offered together with heterospecific females. We found that mate preference for heterospecific females exists in Brazilian T. evansi, suggesting that the preference for heterospecific females is not a lack of evolved premating isolation with an allopatric species. We found that T. evansi males showed lower propensity to mate with heterospecific females when alternative females were non-kin in the two iso-female lines collected from Brazil. However, the effect of relatedness on male mate preference was not significant. We discuss alternative hypotheses explaining why T. evansi males prefer to mate with T. urticae females.

Incomplete premating and postmating reproductive barriers between two parapatric populations of a social spider mite.

Closely related species with overlapping distributions often show premating reproductive barriers to avoid hybridization. Stigmaeopsis miscanthi (Saito) is a social spider mite infesting Chinese silver grass, and the species consists of two parapatric groups with frequent contacts within the contact zone. They differ in male-male aggressiveness, male morphology, female diapause traits, and life history parameters. There is incomplete but strong post-mating reproductive isolation between the two groups, and their DNA sequences are slightly different, suggesting that they diverged recently. In this study, we investigated premating reproductive barriers. We found that females from different groups frequently shared nest webs, indicating no barriers in the phase of nest establishment. However, inside nests, males from either group showed less courtship behaviour to females of the other group and they copulated less frequently with them when compared to females of the same group. However, the premating reproductive barrier was incomplete and asymmetric. Females of one group frequently resisted courtship by males from the other group, but females of the other group did not. We conclude that some gene flow may occur in the contact zone between the two groups.

Association of plasma concentration of 4ß-hydroxycholesterol with CYP3A5 polymorphism and plasma concentration of indoxyl sulfate in stable kidney transplant recipients.

Several studies have shown that renal failure decreases CYP3A activity and that uremic toxins may play a role via transcriptional or translational modifications of cytochrome P450 (P450) enzymes and direct inhibition of P450-mediated metabolism. In this study, we evaluated the relationship between CYP3A activity (using plasma concentration of 4ß-hydroxycholesterol as a biomarker) and clinical characteristics including plasma concentrations of indoxyl sulfate (3-INDS) and indole-3-acetic acid (3-IAA) in stable kidney transplant recipients. Forty-five Japanese kidney transplant recipients who underwent transplantation more than 90 days prior to the study were included. Morning blood samples were collected and plasma concentrations of 4ß-hydroxycholesterol, 3-INDS, and 3-IAA were measured. Plasma concentrations of 4ß-hydroxycholesterol were 57.1 ± 11.2, 42.1 ± 11.8, and 34.5 ± 7.3 ng/ml in recipients with CYP3A5*1/*1 (n = 5), *1/*3 (n = 15), and *3/*3 (n = 25) genotypes, respectively, with significant differences between three genotypes. A significant correlation was observed between plasma concentrations of 4ß-hydroxycholesterol and 3-INDS but not 3-IAA. Multiple regression analysis identified the number of CYP3A5*3 alleles in genotype, plasma concentration of 3-INDS, and body weight as independent variables associated with plasma concentration of 4ß-hydroxycholesterol. In conclusion, these results suggest that CYP3A5 polymorphism and plasma concentration of 3-INDS may account for the interindividual variability of CYP3A activity, and that plasma concentration of 3-INDS may partially explain the gap in CYP3A activity that cannot be explained by genetic contribution in patients with renal failure.

Significant decrease in plasma N-acetyl-seryl-aspartyl-lysyl-proline level in patients with end stage renal disease after kidney transplantation.

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous peptide released from its precursor (thymosin-ß4) by prolyl oligopeptidase. AcSDKP is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. AcSDKP has been shown to be a potent angiogenic factor and to suppress renal fibroblast proliferation. Impairment of renal function has been suggested to have a significant impact on plasma AcSDKP level. The aim of this study was to assess whether improvement of renal function after kidney transplantation has an impact on plasma AcSDKP-like immunoreactive substance (IS) level. Fourteen patients with end stage renal disease (ESRD) who were scheduled to undergo the first kidney allograft transplantation were enrolled. Plasma AcSDKP-IS levels were measured before and 3, 7, 10, 14, 21, 30, 60 and 90 d after kidney transplantation. Plasma AcSDKP-IS level decreased significantly from day 3 after kidney transplantation compared to before kidney transplantation. Creatinine clearance increased significantly from day 7 after kidney transplantation. A significant negative correlation was observed between creatinine clearance and plasma AcSDKP-IS level from before transplantation to 90 d after kidney transplantation. Stepwise multiple regression analysis identified creatinine clearance as the only significant independent factor associated with plasma AcSDKP-IS levels. These results suggest that recovery of kidney function after kidney transplantation may lead to a decrease in plasma AcSDKP level in patients with ESRD, and that plasma AcSDKP level may depend largely on renal function.

Neoseiulus paspalivorus, a predator from coconut, as a candidate for controlling dry bulb mites infesting stored tulip bulbs.

The dry bulb mite, Aceria tulipae, is the most important pest of stored tulip bulbs in The Netherlands. This tiny, eriophyoid mite hides in the narrow space between scales in the interior of the bulb. To achieve biological control of this hidden pest, candidate predators small enough to move in between the bulb scales are required. Earlier experiments have shown this potential for the phytoseiid mite, Neoseiulus cucumeris, but only after the bulbs were exposed to ethylene, a plant hormone that causes a slight increase in the distance between tulip bulb scales, just sufficient to allow this predator to reach the interior part of the bulb. Applying ethylene, however, is not an option in practice because it causes malformation of tulip flowers. In fact, to prevent this cosmetic damage, bulb growers ventilate rooms where tulip bulbs are stored, thereby removing ethylene produced by the bulbs (e.g. in response to mite or fungus infestation). Recently, studies on the role of predatory mites in controlling another eriophyoid mite on coconuts led to the discovery of an exceptionally small phytoseiid mite, Neoseiulus paspalivorus. This predator is able to move under the perianth of coconuts where coconut mites feed on meristematic tissue of the fruit. This discovery prompted us to test N. paspalivorus for its ability to control A. tulipae on tulip bulbs under storage conditions (ventilated rooms with bulbs in open boxes; 23 °C; storage period June-October). Using destructive sampling we monitored predator and prey populations in two series of replicated experiments, one at a high initial level of dry bulb mite infestation, late in the storage period, and another at a low initial dry bulb mite infestation, halfway the storage period. The first and the second series involved treatment with N. paspalivorus and a control experiment, but the second series had an additional treatment in which the predator N. cucumeris was released. Taking the two series of experiments together we found that N. paspalivorus controlled the populations of dry bulb mites both on the outer scale of the bulbs as well as in the interior part of the bulbs, whereas N. cucumeris significantly reduced the population of dry bulb mites on the outer scale, but not in the interior part of the bulb. Moreover, N. paspalivorus was found predominantly inside the bulb, whereas N. cucumeris was only found on the outer scale, thereby confirming our hypothesis that the small size of N. paspalivorus facilitates access to the interior of the bulbs. We argue that N. paspalivorus is a promising candidate for the biological control of dry bulb mites on tulip bulbs under storage conditions in the Netherlands.

Significant increase in plasma 4ß-hydroxycholesterol concentration in patients after kidney transplantation.

Several previous studies have shown that renal failure decreases not only renal elimination but also metabolic clearance of drugs, particularly those metabolized by CYP3A. However, whether recovery of renal function results in recovery of hepatic CYP3A activity remains unknown. In this study, we evaluated the effect of renal function on CYP3A activity after kidney transplantation in patients with end-stage renal disease (ESRD) by measuring the change in CYP3A activity using plasma concentration of 4ß-hydroxycholesterol as a biomarker. The study enrolled 13 patients with ESRD who underwent the first kidney allograft transplantation. Morning blood samples were collected before and 3, 7, 10, 14, 21, 30, 60, 90, 120, 150 and 180 days after kidney transplantation. Plasma concentration of 4ß-hydroxycholesterol was measured using GC-MS. Compared with before kidney transplantation, creatinine clearance increased significantly from day 3 after kidney transplantation and stabilized thereafter. Plasma concentration of 4ß-hydroxycholesterol was elevated significantly on days 90 and 180 after kidney transplantation. In conclusion, this study suggests the recovery of CYP3A activity with improvement in renal function after kidney transplantation in patients with ESRD.

Development and clinical application of an enzyme immunoassay for the determination of midregional proadrenomedullin.

Adrenomedullin (ADM) is a 52-amino acid peptide with a variety of physiologic functions such as immunomodulating activity, direct bactericidal activity, maintenance of renal homeostasis, and vasodilatory activity. Midregional proADM (MR-proADM) is derived from a larger 185-amino acid precursor peptide, prepro-adrenomedulin (preproADM), by posttranslational processing. It is suggested to be co-synthesized with ADM in equimolar amounts and has the advantages over ADM in having a longer half-life, no bioactivity, and no binding to protein. Therefore, MR-proADM serves as a surrogate for ADM secretion. In this study, we attempted to develop an enzyme immunoassay (EIA) for quantifying MR-proADM-like immunoreactive substance (IS), which is applicable for monitoring plasma MR-proADM levels. By using ß-d-galactosidase-labeled preproADM(83-94) as a marker antigen, anti-rabbit IgG-coated immunoplate as a bound/free separator, and 4-methylumbelliferyl-ß-d-galactopyranoside as a fluorogenic substrate, a sensitive and specific EIA was developed for the quantification of MR-proADM-IS in human plasma. The lower limit of quantification was 0.032 pmol/well, and the steep competitive inhibition EIA calibration curve obtained was linear between 0.16 and 10 nmol/L. By using human plasma samples containing 0.2 and 2.0 nmol/L of MR-proADM, the interassay coefficients of variation (reproducibility) were 10.78% and 8.83%, respectively, and intraassay coefficients were 3.91% and 7.81%. Plasma MR-proADM-IS level was significantly higher in patients with chronic renal failure (1.39 ± 0.50 nmol/L) compared with healthy subjects (0.19 ± 0.07 nmol/L). These results suggest that our EIA may be useful to evaluate plasma MR-proADM levels as a biomarker in various clinical settings.

Steely enzymes are involved in prestalk and prespore pattern formation.

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of SteelyA enzyme, controls Dictyostelium spore maturation. Since the expression of stlA split the in early and terminal stages, we cannot exclude the possibility that MPBD regulates spore differentiation from the early stage by creating a bias between the cells. 1-(3,5-Dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-on (DIF-1), a product of SteelyB, was identified as the major stalk cell inducer by in vitro assay, but in vivo assay revealed that DIF-1 induces only prestalkB (pstB) and prestalkO (pstO) cells and, that the major prestalkA (pstA) cells differentiated without DIF-1. In order to determine mechanism of polyketide regulated pattern formation, we examined the spatial expression patterns of prestalk and prespore markers in stlA and stlB knockout mutants. We found that MPBD regulates spore maturation at the culmination stage. We also found that the stlA and stlB double-knockout mutant lost pstA marker gene expression.

Life history differences between two forms of the social spider mite, Stigmaeopsis miscanthi.

The two forms of Stigmaeopsis miscanthi (Saito) (called LW and HG) differ in their levels of male-to-male aggression, differ in their diapause attributes, and morphologically in the male leg I armor. Furthermore, a type of reproductive isolation exists between the forms, although some one-way compatibility is maintained. Here we looked for any differences in life history traits between the two forms under experimental conditions. The intrinsic rate of natural increase (r(m)) of the LW form was lower than that of the HG form, the difference seemingly caused by the developmental speed of the latter. Although the developmental speeds of HG under several temperature regimens were significantly higher than those of LW, the low development threshold temperatures of the two forms were almost identical. This study addresses why a significant difference in r(m) exists in relation to other behavior and physiological (diapause) adaptations.

Asymmetry in male lethal fight between parapatric forms of a social spider mite.

Closely related species often show adjacent geographic distributions, albeit with some overlap. This contiguity is thought to result from secondary contact between (spatially separated) diverging groups or from parapatric speciation. Fights between males of closely related species will affect their chance to mate with females of the other species, which in turn may promote their spatial segregation and drive their speciation. Stigmaeopsis miscanthi is a social spider mite that lives in a group within self-woven nests on leaves of Chinese silver grass. This mite shows lethal male-male fight as a means to maintain a harem, and has two forms showing differences in the levels of male-male aggression, diapause intensity in females and the relative length of the first to third legs. The two forms show parapatric distributions. We found that males of one form readily engage themselves in lethal fight with males of the other form, thereby acquiring the nests and gaining access to females of this other form. Males of the aggressive form tend to win the fights with males of the other form. Their first legs are longer which may provide them with a better weapon and which also indicate a larger body width. However, another determinant of who wins the fight is the length of the third legs which can be a proxy for body length. Based on these results, we hypothesize that male killing behavior is one of the mechanisms maintaining parapatry (instead of sympatry) of the two spider mite forms apart from difference in diapause attributes.

Alternative phenotypes of male mating behaviour in the two-spotted spider mite.

Severe intraspecific competition for mates selects for aggressive individuals but may also lead to the evolution of alternative phenotypes that do not act aggressively, yet manage to acquire matings. The two-spotted spider mite, Tetranychus urticae, shows male mate-guarding behaviour and male-male combat for available females. This may provide opportunity for weaker males to avoid fighting by adopting alternative mating behaviour such as sneaker or satellite tactics as observed in other animals. We investigated male precopulatory behaviour in the two-spotted spider mite by means of video-techniques and found three types of male mating behaviour: territorial, sneaker and opportunistic. Territorial and sneaker males associate with female teleiochrysales and spend much time guarding them. Territorial males are easily disturbed by rival males and engage themselves in fights with them. However, sneaker males are not at all disturbed by rival males, never engage in fights and, strikingly, never face attack by territorial males. Opportunistic males wander around in search of females that are in the teleiochrysalis stage but very close to or at emergence. To quickly classify any given mate-guarding male as territorial or sneaker we developed a method based on the instantaneous response of males to disturbance by a live male mounted on top of a brush. We tested this method against the response of the same males to natural disturbance by two or three other males. Because this method proved to be successful, we used it to collect territorial and sneaker males, and subjected them to morphological analysis to assess whether the various behavioural phenotypes are associated with different morphological characters. However, we found no statistical differences between territorial and sneaker males, concerning the length of the first legs, the stylets, the pedipalps or the body.