Invasion and spread of the neotropical leafhopper Curtara insularis (Hemiptera: Cicadellidae) in Africa and North America and the role of high-altitude windborne migration in invasive insects.

Invasive insects threaten ecosystem stability, public health, and food security. Documenting newly invasive species and understanding how they reach into new territories, establish populations, and interact with other species remain vitally important. Here, we report on the invasion of the South American leafhopper, Curtara insularis into Africa, where it has established populations in Ghana, encroaching inland at least 350 km off the coast. Importantly, 80% of the specimens collected were intercepted between 160 and 190 m above ground. Further, the fraction of this species among all insects collected was also higher at altitude, demonstrating its propensity to engage in high-altitude windborne dispersal. Its aerial densities at altitude translate into millions of migrants/km over a year, representing massive propagule pressure. Given the predominant south-westerly winds, these sightings suggest an introduction of C. insularis into at least one of the Gulf of Guinea ports. To assess the contribution of windborne dispersal to its spread in a new territory, we examine records of C. insularis range-expansion in the USA. Reported first in 2004 from central Florida, it reached north Florida (Panhandle) by 2008-2011 and subsequently spread across the southeastern and south-central US. Its expansion fits a "diffusion-like" process with 200-300 km long "annual displacement steps"-a pattern consistent with autonomous dispersal rather than vehicular transport. Most "steps" are consistent with common wind trajectories from the nearest documented population, assuming 2-8 hours of wind-assisted flight at altitude. Curtara insularis has been intercepted at US ports and on trucks. Thus, it uses multiple dispersal modalities, yet its rapid overland spread is better explained by its massive propagule pressure linked with its high-altitude windborne dispersal. We propose that high-altitude windborne dispersal is common yet under-appreciated in invasive insect species.

Wind-assisted high-altitude dispersal of mosquitoes and other insects in East Africa.

Knowledge of insect dispersal is relevant to the control of agricultural pests, vector-borne transmission of human and veterinary pathogens, and insect biodiversity. Previous studies in a malaria endemic area of the Sahel region in West Africa revealed high-altitude, long-distance migration of insects and various mosquito species. The objective of the current study was to assess whether similar behavior is exhibited by mosquitoes and other insects around the Lake Victoria basin region of Kenya in East Africa. Insects were sampled monthly from dusk to dawn over 1 year using sticky nets suspended on a tethered helium-filled balloon. A total of 17,883 insects were caught on nets tethered at 90, 120, and 160 m above ground level; 818 insects were caught in control nets. Small insects (<0.5 cm, n = 15,250) were predominant regardless of height compared with large insects (>0.5 cm, n = 2,334) and mosquitoes (n = 299). Seven orders were identified; dipteran was the most common. Barcoding molecular assays of 184 mosquitoes identified 7 genera, with Culex being the most common (65.8%) and Anopheles being the least common (5.4%). The survival rate of mosquitoes, experimentally exposed to high-altitude overnight, was significantly lower than controls maintained in the laboratory (19% vs. 85%). There were no significant differences in mosquito survival and oviposition rate according to capture height. These data suggest that windborne dispersal activity of mosquito vectors of malaria and other diseases occurs on a broad scale in sub-Saharan Africa.

Isotopic evidence that aestivation allows malaria mosquitoes to persist through the dry season in the Sahel.

Data suggest that the malaria vector mosquito Anopheles coluzzii persists during the dry season in the Sahel through a dormancy mechanism known as aestivation; however, the contribution of aestivation compared with alternative strategies such as migration is unknown. Here we marked larval Anopheles mosquitoes in two Sahelian villages in Mali using deuterium (2H) to assess the contribution of aestivation to persistence of mosquitoes through the seven-month dry season. After an initial enrichment period, 33% of An. coluzzii mosquitoes were strongly marked. Seven months following enrichment, multiple analysis methods supported the ongoing presence of marked mosquitoes, compatible with the prediction that the fraction of marked mosquitoes should remain stable throughout the dry season if local aestivation is occurring. The results suggest that aestivation is a major persistence mechanism of An. coluzzii in the Sahel, contributing at least 20% of the adults at the onset of rains. This persistence strategy could influence mosquito control and malaria elimination campaigns.

The Effects of High-Altitude Windborne Migration on Survival, Oviposition, and Blood-Feeding of the African Malaria Mosquito, Anopheles gambiae s.l. (Diptera: Culicidae).

Recent results of high-altitude windborne mosquito migration raised questions about the viability of these mosquitoes despite ample evidence that many insect species, including other dipterans, have been known to migrate regularly over tens or hundreds of kilometers on high-altitude winds and retain their viability. To address these concerns, we subjected wild Anopheles gambiae s.l. Giles mosquitoes to a high-altitude survival assay, followed by oviposition (egg laying) and blood feeding assays. Despite carrying out the survival assay under exceptionally harsh conditions that probably provide the lowest survival potential following high altitude flight, a high proportion of the mosquitoes survived for 6- and even 11-h assay durations at 120- to 250-m altitudes. Minimal differences in egg laying success were noted between mosquitoes exposed to high altitude survival assay and those kept near the ground. Similarly, minimal differences were found in the female's ability to take an additional bloodmeal after oviposition between these groups. We conclude that similar to other high-altitude migrating insects, mosquitoes are able to withstand extended high-altitude flight and subsequently reproduce and transmit pathogens by blood feeding on new hosts.

Diversity, dynamics, direction, and magnitude of high-altitude migrating insects in the Sahel.

Long-distance migration of insects impacts food security, public health, and conservation-issues that are especially significant in Africa. Windborne migration is a key strategy enabling exploitation of ephemeral havens such as the Sahel, however, its knowledge remains sparse. In this first cross-season investigation (3 years) of the aerial fauna over Africa, we sampled insects flying 40-290 m above ground in Mali, using nets mounted on tethered helium-filled balloons. Nearly half a million insects were caught, representing at least 100 families from thirteen orders. Control nets confirmed that the insects were captured at altitude. Thirteen ecologically and phylogenetically diverse species were studied in detail. Migration of all species peaked during the wet season every year across localities, suggesting regular migrations. Species differed in flight altitude, seasonality, and associated weather conditions. All taxa exhibited frequent flights on southerly winds, accounting for the recolonization of the Sahel from southern source populations. "Return" southward movement occurred in most taxa. Estimates of the seasonal number of migrants per species crossing Mali at latitude 14°N were in the trillions, and the nightly distances traversed reached hundreds of kilometers. The magnitude and diversity of windborne insect migration highlight its importance and impacts on Sahelian and neighboring ecosystems.

Quantifying flight aptitude variation in wild Anopheles gambiae in order to identify long-distance migrants.

BACKGROUND: In the West African Sahel, mosquito reproduction is halted during the 5-7 month-long dry season, due to the absence of surface waters required for larval development. However, recent studies have suggested that both Anopheles gambiae sensu stricto (s.s.) and Anopheles arabiensis repopulate this region via migration from distant locations where larval sites are perennial. Anopheles coluzzii engages in more regional migration, presumably within the Sahel, following shifting resources correlating with the ever-changing patterns of Sahelian rainfall. Understanding mosquito migration is key to controlling malaria-a disease that continues to claim more than 400,000 lives annually, especially those of African children. Using tethered flight data of wild mosquitoes, the distribution of flight parameters were evaluated as indicators of long-range migrants versus appetitive flyers, and the species specific seasonal differences and gonotrophic states compared between two flight activity modalities. Morphometrical differences were evaluated in the wings of mosquitoes exhibiting high flight activity (HFA) vs. low flight activity (LFA). METHODS: A novel tethered-flight assay was used to characterize flight in the three primary malaria vectors- An. arabiensis, An. coluzzii and An. gambiae s.s. The flights of tethered wild mosquitoes were audio-recorded from 21:00 h to 05:00 h in the following morning and three flight aptitude indices were examined: total flight duration, longest flight bout, and the number of flight bouts during the assay. RESULTS: The distributions of all flight indices were strongly skewed to the right, indicating that the population consisted of a majority of low-flight activity (LFA) mosquitoes and a minority of high-flight activity (HFA) mosquitoes. The median total flight was 586 s and the maximum value was 16,110 s (~ 4.5 h). In accordance with recent results, flight aptitude peaked in the wet season, and was higher in gravid females than in non-blood-fed females. Flight aptitude was also found to be higher in An. coluzzii compared to An. arabiensis, with intermediate values in An. gambiae s.s., but displaying no statistical difference. Evaluating differences in wing size and shape between LFA individuals and HFA ones, the wing size of HFA An. coluzzii was larger than that of LFAs during the wet season-its length was wider than predicted by allometry alone, indicating a change in wing shape. No statistically significant differences were found in the wing size/shape of An. gambiae s.s. or An. arabiensis. CONCLUSIONS: The partial agreement between the tethered flight results and recent results based on aerial sampling of these species suggest a degree of discrimination between appetitive flyers and long-distance migrants although identifying HFAs as long-distance migrants is not recommended without further investigation.

Windborne long-distance migration of malaria mosquitoes in the Sahel.

Over the past two decades efforts to control malaria have halved the number of cases globally, yet burdens remain high in much of Africa and the elimination of malaria has not been achieved even in areas where extreme reductions have been sustained, such as South Africa1,2. Studies seeking to understand the paradoxical persistence of malaria in areas in which surface water is absent for 3-8 months of the year have suggested that some species of Anopheles mosquito use long-distance migration3. Here we confirm this hypothesis through aerial sampling of mosquitoes at 40-290 m above ground level and provide-to our knowledge-the first evidence of windborne migration of African malaria vectors, and consequently of the pathogens that they transmit. Ten species, including the primary malaria vector Anopheles coluzzii, were identified among 235 anopheline mosquitoes that were captured during 617 nocturnal aerial collections in the Sahel of Mali. Notably, females accounted for more than 80% of all of the mosquitoes that we collected. Of these, 90% had taken a blood meal before their migration, which implies that pathogens are probably transported over long distances by migrating females. The likelihood of capturing Anopheles species increased with altitude (the height of the sampling panel above ground level) and during the wet seasons, but variation between years and localities was minimal. Simulated trajectories of mosquito flights indicated that there would be mean nightly displacements of up to 300 km for 9-h flight durations. Annually, the estimated numbers of mosquitoes at altitude that cross a 100-km line perpendicular to the prevailing wind direction included 81,000 Anopheles gambiae sensu stricto, 6 million A. coluzzii and 44 million Anopheles squamosus. These results provide compelling evidence that millions of malaria vectors that have previously fed on blood frequently migrate over hundreds of kilometres, and thus almost certainly spread malaria over these distances. The successful elimination of malaria may therefore depend on whether the sources of migrant vectors can be identified and controlled.

Investigation of the seasonal microbiome of Anopheles coluzzii mosquitoes in Mali.

The poorly understood mechanisms of dry season persistence of Anopheles spp. mosquitoes through the dry season in Africa remain a critical gap in our knowledge of Plasmodium disease transmission. While it is thought that adult mosquitoes remain in a dormant state throughout this seven-month dry season, the nature of this state remains unknown and has largely not been recapitulated in laboratory settings. To elucidate possible connections of this state with microbial composition, the whole body microbiomes of adult mosquitoes in the dry and wet seasons in two locations of Mali with varying water availability were compared by sequencing the 16S ribosomal RNA gene. These locations were a village near the Niger River with year-round water sources (N'Gabakoro, "riparian"), and a typical Sahelian area with highly seasonal breeding sites (Thierola Area, "Sahelian"). The 16S bacterial data consisted of 2057 sequence variants in 426 genera across 184 families. From these data, we found several compositional differences that were seasonally and spatially linked. Counter to our initial hypothesis, there were more pronounced seasonal differences in the bacterial microbiome of riparian, rather than Sahelian areas. These seasonal shifts were primarily in Ralstonia, Sphingorhabdus, and Duganella spp. bacteria that are usually soil and water-associated, indicating these changes may be from bacteria acquired in the larval environment, rather than adulthood. In Sahelian dry season mosquitoes, there was a unique intracellular bacteria, Anaplasma, which likely was acquired through non-human blood feeding. Cytochrome B analysis of blood meals showed greater heterogeneity in host choice of An. coluzzii independent of season in the Thierola area compared to N'Gabakoro (77.5% vs. 94.6% human-origin blood meal, respectively), indicating a relaxation of anthropophily. Overall, this exploratory study provides valuable indications of spatial and seasonal differences in bacterial composition which help refine this difficult to study state.

Tracing the origin of the early wet-season Anopheles coluzzii in the Sahel.

In arid environments, the source of the malaria mosquito populations that re-establish soon after first rains remains a puzzle and alternative explanations have been proposed. Using genetic data, we evaluated whether the early rainy season (RS) population of Anopheles coluzzii is descended from the preceding late RS generation at the same locality, consistent with dry season (DS) dormancy (aestivation), or from migrants from distant locations. Distinct predictions derived from these two hypotheses were assessed, based on variation in 738 SNPs in eleven A. coluzzii samples, including seven samples spanning 2 years in a Sahelian village. As predicted by the "local origin under aestivation hypothesis," temporal samples from the late RS and those collected after the first rain of the following RS were clustered together, while larger genetic distances were found among samples spanning the RS. Likewise, multilocus genotype composition of samples from the end of the RS was similar across samples until the following RS, unlike samples that spanned the RS. Consistent with reproductive arrest during the DS, no genetic drift was detected between samples taken over that period, despite encompassing extreme population minima, whereas it was detected between samples spanning the RS. Accordingly, the variance in allele frequency increased with time over the RS, but not over the DS. However, not all the results agreed with aestivation. Large genetic distances separated samples taken a year apart, and during the first year, within-sample genetic diversity declined and increased back during the late RS, suggesting a bottleneck followed by migration. The decline of genetic diversity followed by a mass distribution of insecticide-treated nets was accompanied by a reduced mosquito density and a rise in the mutation conferring resistance to pyrethroids, indicating a bottleneck due to insecticidal selection. Overall, our results support aestivation in A. coluzzii during the DS that is accompanied by long-distance migration in the late RS.

Desiccation tolerance in Anopheles coluzzii: the effects of spiracle size and cuticular hydrocarbons.

The African malaria mosquitoes Anopheles gambiae and Anopheles coluzzii range over forests and arid areas, where they withstand dry spells and months-long dry seasons, suggesting variation in their desiccation tolerance. We subjected a laboratory colony (G3) and wild Sahelian mosquitoes during the rainy and dry seasons to desiccation assays. The thoracic spiracles and amount and composition of cuticular hydrocarbons (CHCs) of individual mosquitoes were measured to determine the effects of these traits on desiccation tolerance. The relative humidity of the assay, body water available, rate of water loss and water content at death accounted for 88% of the variation in desiccation tolerance. Spiracle size did not affect the rate of water loss or desiccation tolerance of the colony mosquitoes, as was the case for the total CHCs. However, six CHCs accounted for 71% of the variation in desiccation tolerance and three accounted for 72% of the variation in the rate of water loss. Wild A. coluzzii exhibited elevated desiccation tolerance during the dry season. During that time, relative thorax and spiracle sizes were smaller than during the rainy season. A smaller spiracle size appeared to increase A. coluzzii's desiccation tolerance, but was not statistically significant. Seasonal changes in CHC composition were detected in Sahelian A. coluzzii Stepwise regression models suggested the effect of particular CHCs on desiccation tolerance. In conclusion, the combination of particular CHCs along with the total amount of CHCs is a primary mechanism conferring desiccation tolerance in A. coluzzii, while variation in spiracle size might be a secondary mechanism.

Seasonal variation in metabolic rate, flight activity and body size of Anopheles gambiae in the Sahel.

Malaria in Africa is vectored primarily by the Anopheles gambiae complex. Although the mechanisms of population persistence during the dry season are not yet known, targeting dry season mosquitoes could provide opportunities for vector control. In the Sahel, it appears likely that M-form A. gambiae survive by aestivation (entering a dormant state). To assess the role of eco-physiological changes associated with dry season survival, we measured body size, flight activity and metabolic rate of wild-caught mosquitoes throughout 1 year in a Sahelian locality, far from permanent water sources, and at a riparian location adjacent to the Niger River. We found significant seasonal variation in body size at both the Sahelian and riparian sites, although the magnitude of the variation was greater in the Sahel. For flight activity, significant seasonality was only observed in the Sahel, with increased flight activity in the wet season when compared with that just prior to and throughout the dry season. Whole-organism metabolic rate was affected by numerous biotic and abiotic factors, and a significant seasonal component was found at both locations. However, assay temperature accounted completely for seasonality at the riparian location, while significant seasonal variation remained after accounting for all measured variables in the Sahel. Interestingly, we did not find that mean metabolic rate was lowest during the dry season at either location, contrary to our expectation that mosquitoes would conserve energy and increase longevity by reducing metabolism during this time. These results indicate that mosquitoes may use mechanisms besides reduced metabolic rate to enable survival during the Sahelian dry season.

Dry season reproductive depression of Anopheles gambiae in the Sahel.

The African malaria mosquito, Anopheles gambiae, is widespread south of the Sahara including in dry savannahs and semi-arid environments where no surface water exists for several months a year. Adults of the M form of An. gambiae persist through the long dry season, when no surface waters are available, by increasing their maximal survival from 4 weeks to 7 months. Dry season diapause (aestivation) presumably underlies this extended survival. Diapause in adult insects is intrinsically linked to depressed reproduction. To determine if reproduction of the Sahelian M form is depressed during the dry season, we assessed seasonal changes in oviposition, egg batch size, and egg development, as well as insemination rate and blood feeding in wild caught mosquitoes. Results from xeric Sahelian and riparian populations were compared. Oviposition response in the Sahelian M form dropped from 70% during the wet season to 20% during the dry season while the mean egg batch size among those that laid eggs fell from 173 to 101. Correspondingly, the fraction of females that exhibited gonotrophic dissociation increased over the dry season from 5% to 45%, while a similar fraction of the population retained developed eggs despite having access to water. This depression in reproduction the Sahelian M form was not caused by a reduced insemination rate. Seasonal variation in these reproductive parameters of the riparian M form population was less extreme and the duration of reproductive depression was shorter. Blood feeding responses did not change with the season in either population. Depressed reproduction during the dry season in the Sahelian M form of An. gambiae provides additional evidence for aestivation and illuminates the physiological processes involved. The differences between the Sahelian and riparian population suggest an adaptive cline in aestivation phenotypes between populations only 130 km apart.

Spatial distribution and male mating success of Anopheles gambiae swarms.

BACKGROUND: Anopheles gambiae mates in flight at particular mating sites over specific landmarks known as swarm markers. The swarms are composed of males; females typically approach a swarm, and leave in copula. This mating aggregation looks like a lek, but appears to lack the component of female choice. To investigate the possible mechanisms promoting the evolution of swarming in this mosquito species, we looked at the variation in mating success between swarms and discussed the factors that structure it in light of the three major lekking models, known as the female preference model, the hotspot model, and the hotshot model. RESULTS: We found substantial variation in swarm size and in mating success between swarms. A strong correlation between swarm size and mating success was observed, and consistent with the hotspot model of lek formation, the per capita mating success of individual males did not increase with swarm size. For the spatial distribution of swarms, our results revealed that some display sites were more attractive to both males and females and that females were more attracted to large swarms. While the swarm markers we recognize help us in localizing swarms, they did not account for the variation in swarm size or in the swarm mating success, suggesting that mosquitoes probably are attracted to these markers, but also perceive and respond to other aspects of the swarming site. CONCLUSIONS: Characterizing the mating system of a species helps understand how this species has evolved and how selective pressures operate on male and female traits. The current study looked at male mating success of An. gambiae and discussed possible factors that account for its variation. We found that swarms of An. gambiae conform to the hotspot model of lek formation. But because swarms may lack the female choice component, we propose that the An. gambiae mating system is a lek-like system that incorporates characteristics pertaining to other mating systems such as scramble mating competition.

Variation in metabolic rate of Anopheles gambiae and A. arabiensis in a Sahelian village.

In the Sahel, the Anopheles gambiae complex consists of Anopheles arabiensis and the M and S molecular forms of A. gambiae sensu stricto. However, the composition of these malaria vectors varies spatially and temporally throughout the region and is thought to be linked to environmental factors such as rainfall, larval site characteristics and duration of the dry season. To examine possible physiological divergence between these taxa, we measured metabolic rates of mosquitoes during the wet season in a Sahelian village in Mali. To our knowledge, this study provides the first measurements of metabolic rates of A. gambiae and A. arabiensis in the field. The mean metabolic rate of A. arabiensis was higher than that of M-form A. gambiae when accounting for the effects of female gonotrophic status, temperature and flight activity. However, after accounting for their difference in body size, no significant difference in metabolic rate was found between these two species (whilst all other factors were found to be significant). Thus, body size may be a key character that has diverged in response to ecological differences between these two species. Alternatively, these species may display additional differences in metabolic rate only during the dry season. Overall, our results indicate that changes in behavior and feeding activity provide an effective mechanism for mosquitoes to reduce their metabolic rate, and provide insight into the possible strategies employed by aestivating individuals during the dry season. We hypothesize that female mosquitoes switch to sugar feeding while in dormancy because of elevated metabolism associated with blood digestion.

3D tracking of mating events in wild swarms of the malaria mosquito Anopheles gambiae.

We describe an automated tracking system that allows us to reconstruct the 3D kinematics of individual mosquitoes in swarms of Anopheles gambiae. The inputs to the tracking system are video streams recorded from a stereo camera system. The tracker uses a two-pass procedure to automatically localize and track mosquitoes within the swarm. A human-in-the-loop step verifies the estimates and connects broken tracks. The tracker performance is illustrated using footage of mating events filmed in Mali in August 2010.

Spatial swarm segregation and reproductive isolation between the molecular forms of Anopheles gambiae.

Anopheles gambiae, the major malaria vector in Africa, can be divided into two subgroups based on genetic and ecological criteria. These two subgroups, termed the M and S molecular forms, are believed to be incipient species. Although they display differences in the ecological niches they occupy in the field, they are often sympatric and readily hybridize in the laboratory to produce viable and fertile offspring. Evidence for assortative mating in the field was recently reported, but the underlying mechanisms awaited discovery. We studied swarming behaviour of the molecular forms and investigated the role of swarm segregation in mediating assortative mating. Molecular identification of 1145 males collected from 68 swarms in Donéguébougou, Mali, over 2 years revealed a strict pattern of spatial segregation, resulting in almost exclusively monotypic swarms with respect to molecular form. We found evidence of clustering of swarms composed of individuals of a single molecular form within the village. Tethered M and S females were introduced into natural swarms of the M form to verify the existence of possible mate recognition operating within-swarm. Both M and S females were inseminated regardless of their form under these conditions, suggesting no within-mate recognition. We argue that our results provide evidence that swarm spatial segregation strongly contributes to reproductive isolation between the molecular forms in Mali. However this does not exclude the possibility of additional mate recognition operating across the range distribution of the forms. We discuss the importance of spatial segregation in the context of possible geographic variation in mechanisms of reproductive isolation.

Structure and dynamics of male swarms of Anopheles gambiae.

Mosquito swarms are poorly understood mating aggregations. In the malaria vector Anopheles gambiae Giles, they are known to depend on environmental conditions, such as the presence of a marker on the ground, and they may be highly relevant to reproductive isolation. We present quantitative measurements of individual An. gambiae positions within swarms from Donéguébougou, Mali, estimated by stereoscopic video image analysis. Results indicate that swarms in this species are approximately spherical, with an unexpectedly high density of individuals close to the swarm centroid. This high density may be the result of individual males maximizing their probability of encountering a female or a product of mosquito orientation through cues within the swarm. Our analysis also suggests a difference in swarm organization between putative incipient species of An. gambiae with increasing numbers of males. This may be related to a difference in marker use between these groups, supporting the hypothesis that swarming behavior is a mechanism of mate recognition and ultimately reproductive isolation.

The distribution of hatching time in Anopheles gambiae.

BACKGROUND: Knowledge of the ecological differences between the molecular forms of Anopheles gambiae and their sibling species, An. arabiensis might lead to understanding their unique contribution to disease transmission and to better vector control as well as to understanding the evolutionary forces that have separated them. METHODS: The distributions of hatching time of eggs of wild An. gambiae and An. arabiensis females were compared in different water types. Early and late hatchers of the S molecular form were compared with respect to their total protein content, sex ratio, development success, developmental time and adult body size. RESULTS: Overall, the distribution of hatching time was strongly skewed to the right, with 89% of the eggs hatching during the second and third day post oviposition, 10% hatching during the next four days and the remaining 1% hatching over the subsequent week. Slight, but significant differences were found between species and between the molecular forms in all water types. Differences in hatching time distribution were also found among water types (in each species and molecular form), suggesting that the eggs change their hatching time in response to chemical factors in the water. Early hatchers were similar to late hatchers except that they developed faster and produced smaller adults than late hatchers. CONCLUSION: Differences in hatching time and speed of development among eggs of the same batch may be adaptive if catastrophic events such as larval site desiccation are not rare and the site's quality is unpredictable. The egg is not passive and its hatching time depends on water factors. Differences in hatching time between species and molecular forms were slight, probably reflecting that conditions in their larval sites are rather similar.