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Ross, P. A.

Publications and source records attributed to Ross, P. A..

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Local adaptation of Aedes aegypti mosquitoes to Wolbachia-induced fitness costs

Aedes aegypti mosquito eggs can remain quiescent for many months before hatching, allowing populations to persist through unfavorable conditions. Aedes aegypti infected with the Wolbachia strain wMel have been released in tropical and subtropical regions for dengue control. wMel reduces the viability of quiescent eggs, but this physiological cost might be expected to evolve in natural mosquito populations that frequently experience stressful conditions. We therefore compared the costs of wMel infection for quiescent egg viability in field-derived and laboratory populations. Quiescent egg viability was highly variable in wMel-infected populations, with greater costs of wMel in field-derived populations. In contrast, there was little variation between matched field-derived and long-term laboratory populations lacking wMel, suggesting that laboratory adaptation does not influence this trait and that differences are due to wMel infection. Comparisons of populations collected a year apart show a decline in costs under laboratory rearing conditions involving a rapid turnover of mosquito generations; this pattern was consistent across populations despite their origin, suggesting adaptation of mosquitoes to the wMel infection under laboratory conditions. Reciprocal crossing experiments confirm that differences in quiescent egg viability were mainly due to the genetic background and not Wolbachia alone. wMel-infected mosquitoes hatching from long-term quiescent eggs showed partial loss of cytoplasmic incompatibility and female infertility, highlighting additional costs of long-term quiescence. Our study provides the first evidence for a shift in Wolbachia phenotypic effects following deliberate field release and establishment and it highlights interactions between Wolbachia infections and local adaptation. The unexpected changes in fitness costs observed here suggest potential tradeoffs with undescribed fitness benefits of the wMel infection.

evolutionary biology↗

Wolbachia inhibits ovarian formation and increases blood avidity in female Aedes aegypti-a novel time-specific reproductive alteration

Wolbachia, a gram-negative endosymbiotic bacterium widespread in arthropods, is well-known for changing the reproduction of its host in ways that increase its rate of spread, but there are also costs to hosts that can reduce this. Here we investigated a novel reproductive alteration of Wolbachia wAlbB on its mosquito host Aedes aegypti, based on studies of mosquito life history traits, ovarian dissections and gene expression assays. We found that an extended period of the larval stage as well as the egg stage, as previously shown, can increase the proportion of Wolbachia-infected females that become infertile; the impact of Wolbachia on infertility therefore accumulated before pupation. We found that ovarian formation was blocked in infertile females, even though infected females had relatively lower Wolbachia densities than fertile infected females. Infertile females also showed a higher frequency of blood feeding following a prior blood meal, indicating that they do not enter a complete gonotrophic cycle. Treatments leading to infertility decreased expression of genes related to reproduction. Our results demonstrate effects related to the causes and consequences of infertile wAlbB-infected Ae. aegypti females with implications for Wolbachia releases and have evolutionary implications for Wolbachia infections in novel hosts.

microbiology↗

Genomic and phenotypic comparisons reveal distinct variants of Wolbachia strain wAlbB

The intracellular bacterium Wolbachia inhibits virus replication and is being harnessed around the world to fight mosquito-borne diseases through releases of mosquitoes carrying the symbiont. Wolbachia strains vary in their ability to invade mosquito populations and suppress viruses in part due to differences in their density within the insect and associated fitness costs. Using whole-genome sequencing, we demonstrate the existence of two variants in wAlbB, a Wolbachia strain being released in natural populations of Aedes aegypti mosquitoes. The two variants display striking differences in genome architecture and gene content. Differences in the presence/absence of 49 genes between variants include genes located in prophage regions and others potentially involved in controlling the symbionts density. Importantly, we show that these genetic differences correlate with variation in wAlbB density and its tolerance to heat stress, suggesting that different wAlbB variants may be better suited for field deployment depending on local environmental conditions. Finally, we found that the wAlbB genome remained stable following its introduction in a Malaysian mosquito population. Our results highlight the need for further genomic and phenotypic characterization of Wolbachia strains in order to inform ongoing Wolbachia-based programmes and improve the selection of optimal strains in future field interventions. ImportanceDengue is a viral disease transmitted by Aedes mosquitoes that threatens around half of the world population. Recent advances in dengue control involve the introduction of Wolbachia bacterial symbionts with antiviral properties into mosquito populations which can lead to dramatic decreases in the incidence of the disease. In light of these promising results, there is a crucial need to better understand the factors affecting the success of such strategies, in particular the choice of Wolbachia strain for field releases and the potential for evolutionary changes. Here we characterized two variants of a Wolbachia strain used for dengue control that differ at the genomic level and in their ability to replicate within the mosquito. We also found no evidence for the evolution of the symbiont within the two years following its deployment in Malaysia. Our results have implications for current and future Wolbachia-based health interventions.

genomics↗

A wMel Wolbachia variant in Aedes aegypti from field-collected Drosophila melanogaster with increased phenotypic stability under heat stress

Mosquito-borne diseases such as dengue, Zika and chikungunya remain a major cause of morbidity and mortality across tropical regions. Population replacement strategies involving the wMel strain of Wolbachia are being used widely to control mosquito-borne diseases transmitted by Aedes aegypti. However, these strategies may be influenced by environmental temperature because wMel is vulnerable to heat stress. wMel infections in their native host Drosophila melanogaster are genetically diverse, but few transinfections of wMel variants have been generated in Ae. aegypti mosquitoes. Here we successfully transferred a wMel variant (termed wMelM) originating from a field-collected D. melanogaster population from Victoria, Australia into Ae. aegypti. The new wMelM variant (clade I) is genetically distinct from the original wMel transinfection (clade III) generated over ten years ago, and there are no genomic differences between wMelM in its original and transinfected host. We compared wMelM with wMel in its effects on host fitness, temperature tolerance, Wolbachia density, vector competence, cytoplasmic incompatibility and maternal transmission under heat stress in a controlled background. wMelM showed a higher heat tolerance than wMel, with stronger cytoplasmic incompatibility and maternal transmission when eggs were exposed to heat stress, likely due to higher overall densities within the mosquito. Both wMel variants had minimal host fitness costs, complete cytoplasmic incompatibility and maternal transmission, and dengue virus blocking under standard laboratory conditions. Our results highlight phenotypic differences between closely related Wolbachia variants. wMelM shows potential as an alternative strain to wMel in dengue control programs in areas with strong seasonal temperature fluctuations.

microbiology↗

A decade of stability for wMel Wolbachia in natural Aedes aegypti populations

Mosquitoes carrying Wolbachia endosymbionts are being released in many countries for arbovirus control. The wMel strain of Wolbachia blocks Aedes-borne virus transmission and can spread throughout mosquito populations by inducing cytoplasmic incompatibility. Aedes aegypti mosquitoes carrying wMel were first released into the field in Cairns, Australia, over a decade ago, and with wider releases have resulted in the near elimination of local dengue transmission. The long-term stability of Wolbachia effects is critical for ongoing disease suppression, requiring tracking of phenotypic and genomic changes in Wolbachia infections following releases. We used a combination of field surveys, phenotypic assessments, and Wolbachia genome sequencing to show that wMel has remained stable in its effects for up to a decade in Australian Ae. aegypti populations. Phenotypic comparisons of wMel-infected and uninfected mosquitoes from near-field and long-term laboratory populations suggest limited changes in the effects of wMel on mosquito fitness. Treating mosquitoes with antibiotics used to cure the wMel infection had limited effects on fitness in the next generation, supporting the use of tetracycline for generating uninfected mosquitoes without off-target effects. wMel has a temporally stable within-host density and continues to induce complete cytoplasmic incompatibility. A comparison of wMel genomes from pre-release (2010) and nine years post-release (2020) populations show few genomic differences and little divergence between release locations, consistent with the lack of phenotypic changes. These results indicate that releases of Wolbachia-infected mosquitoes for population replacement are likely to be effective for many years, but ongoing monitoring remains important to track potential evolutionary changes. Author summaryWolbachia are endosymbionts that can block the transmission of arboviruses by mosquitoes. Aedes aegypti mosquitoes carrying the wMel strain of Wolbachia have been released in population replacement interventions, which aim to establish wMel in mosquito populations, thereby reducing their ability to spread disease. Wolbachia population replacement programs began only a decade ago, raising uncertainty about their long-term effectiveness. Here we provide a comprehensive assessment of the long-term stability of wMel from the very first Wolbachia population replacement release. We show that there is no evidence for changes in the phenotypic effects of wMel in mosquitoes, and confirm that the wMel genome has changed very little in the decade since field releases began. wMel remains at high levels within mosquitoes, suggesting that its ability to block virus transmission has been retained. Our data provides confidence that Wolbachia population replacement releases will provide ongoing protection against arbovirus transmission.

evolutionary biology↗

A wAlbB Wolbachia transinfection displays stable phenotypic effects across divergent Aedes aegypti mosquito backgrounds

Aedes mosquitoes harboring intracellular Wolbachia bacteria are being released in arbovirus and mosquito control programs. With releases taking place around the world, understanding the contribution of host variation to Wolbachia phenotype is crucial. We generated a Wolbachia transinfection (wAlbBQ) in Aedes aegypti and performed backcrossing to introduce the infection into Australian or Malaysian nuclear backgrounds. Whole Wolbachia genome sequencing shows that the wAlbBQ transinfection is near-identical to the reference wAlbB genome, suggesting few changes since the infection was first introduced to Ae. aegypti over 15 years ago. However, these sequences were distinct from other available wAlbB genome sequences, highlighting the potential diversity of wAlbB in natural Ae. albopictus populations. Phenotypic comparisons demonstrate effects of wAlbB infection on egg hatch and nuclear background on fecundity and body size, but no interactions between wAlbB infection and nuclear background for any trait. The wAlbB infection was stable at high temperatures and showed perfect maternal transmission and cytoplasmic incompatibility regardless of host background. Our results demonstrate the stability of wAlbB across host backgrounds and point to its long-term effectiveness for controlling arbovirus transmission and mosquito populations.

evolutionary biology↗

Strong attraction of male mosquitoes (Aedes aegypti) to humans

Female Aedes aegypti mosquitoes integrate multiple sensory cues to locate human hosts for blood meals. While male mosquitoes do not blood feed, male Ae. aegypti swarm around and land on humans in nature. Basrur et al. (2020) generated male Ae. aegypti lacking the fruitless gene and discovered that they gained strong attraction to humans, similar to female mosquitoes. The authors assume that host-seeking is a female-specific trait. However, all experiments were performed under confined laboratory conditions which are unable to detect long-range attraction. We used semi-field experiments to demonstrate robust attraction of male Ae. aegypti to humans. Our observations refute a key assumption of Basrur et al. (2020) and raise questions around conditions under which fruitless prevents male host-seeking. Male mosquito attraction to humans is likely to be important for mating success in wild populations and its basis should be further explored.

animal behavior and cognition↗

Infertility and fecundity loss of Wolbachia-infected Aedes aegypti hatched from quiescent eggs is expected to alter invasion dynamics

The endosymbiotic bacterium Wolbachia shows viral blockage in its mosquito host, leading to its use in arboviral disease control. Releases with Wolbachia strains wMel and wAlbB infecting Aedes aegypti have taken place in several countries. Mosquito egg survival is a key factor influencing population persistence and this trait is also important when eggs are stored prior to releases. We therefore tested the viability of mosquitoes derived from Wolbachia wMel and wAlbB-infected as well as uninfected eggs after long-term storage under diurnal temperature cycles of 11-19{degrees}C and 22-30{degrees}C. Eggs stored at 11-19{degrees}C had higher hatch proportions than those stored at 22-30{degrees}C. Adult Wolbachia density declined when they emerged from eggs stored for longer, which was associated with incomplete cytoplasmic incompatibility (CI) when wMel-infected males were crossed with uninfected females. wAlbB-infected males continued to show complete CI. Females from stored eggs at both temperatures continued to show perfect maternal transmission of Wolbachia, but storage reduced the fecundity of both wMel and wAlbB-infected females relative to uninfected mosquitoes. Furthermore, we found a very strong negative impact of the wAlbB infection on the fertility of females stored at 22-30{degrees}C, with almost 80% of females hatching after 11 weeks of storage being infertile. Our findings provide guidance for storing Wolbachia-infected Ae. aegypti eggs to ensure high fitness adult mosquitoes for release. Importantly, they also highlight the likely impact of egg quiescence on the population dynamics of Wolbachia-infected populations in the field, and the potential for Wolbachia to suppress mosquito populations through cumulative fitness costs across warm and dry periods, with expected effects on dengue transmission.

evolutionary biology↗

Stable phenotypic effects of an unstable deleterious Wolbachia infection

Wolbachia are being used to reduce dengue transmission by Aedes aegypti mosquitoes around the world. To date releases have mostly involved Wolbachia strains with limited fitness effects but strains with larger fitness costs could be used to suppress mosquito populations. However, such infections are expected to evolve towards decreased deleterious effects. Here we investigate potential evolutionary changes in the wMelPop infection transferred from Drosophila melanogaster to Aedes aegypti more than ten years (~120 generations) ago. We show that most deleterious effects of this infection have persisted despite strong selection to ameliorate them. The wMelPop infection is difficult to maintain in laboratory colonies, likely due to the persistent deleterious effects coupled with occasional maternal transmission leakage. Furthermore, female mosquitoes can be scored incorrectly as infected due to transmission of Wolbachia through mating. Infection loss in colonies was not associated with evolutionary changes in the nuclear background. These findings suggest that Wolbachia transinfections with deleterious effects may have stable phenotypes which could ensure their long-term effectiveness if released in natural populations to reduce population size. Author summaryMosquitoes infected with Wolbachia bacteria are being deployed into the field where they can suppress mosquito populations and reduce dengue transmission. These programs rely on the use of Wolbachia strains that have desirable phenotypes, which can include deleterious fitness effects, reproductive manipulation and virus blocking. However, theory predicts that Wolbachia will evolve to become less costly to their hosts, reducing the effectiveness of these programs. We investigate the potential for evolutionary changes by performing a comprehensive phenotypic analysis of a deleterious Wolbachia strain, wMelPop, that was introduced to Aedes aegypti mosquitoes from Drosophila over ten years ago. In contrast to theoretical expectations and research from Drosophila, our results suggest that Wolbachia strains with deleterious effects may have stable phenotypes, ensuring their long-term effectiveness if released into natural populations.

evolutionary biology↗

An elusive endosymbiont: Does Wolbachia occur naturally in Aedes aegypti?

Wolbachia are maternally-inherited endosymbiotic bacteria found within many insect species. Aedes mosquitoes experimentally infected with Wolbachia are being released into the field for Aedes-borne disease control. These Wolbachia infections induce cytoplasmic incompatibility which is used to suppress populations through incompatible matings or replace populations through the reproductive advantage provided by this mechanism. However the presence of naturally-occurring Wolbachia in target populations could interfere with both population replacement and suppression programs depending on the compatibility patterns between strains. Aedes aegypti were thought to not harbor Wolbachia naturally but several recent studies have detected Wolbachia in natural populations of this mosquito. We therefore review the evidence for natural Wolbachia infections in Ae. aegypti to date and discuss limitations of these studies. We draw on research from other mosquito species to outline the potential implications of natural Wolbachia infections in Ae. aegypti for disease control. To validate previous reports, we obtained a laboratory population of Ae. aegypti from New Mexico, USA, that harbors a natural Wolbachia infection, and we conducted field surveys in Kuala Lumpur, Malaysia where a natural Wolbachia infection has also been reported. However, we were unable to detect Wolbachia infection in both the laboratory and field populations. Because the presence of naturally-occurring Wolbachia in Ae. aegypti could have profound implications for Wolbachia-based disease control programs, it is important to continue to accurately assess the Wolbachia status of target Aedes populations.

ecology↗

A LAMP assay for the rapid and robust assessment of Wolbachia infection in Aedes aegypti under field and laboratory conditions

With Wolbachia-based arbovirus control programs being scaled and operationalised around the world, cost effective and reliable detection of Wolbachia in field samples and laboratory stocks is essential for quality control. Here we validate a modified loop-mediated isothermal amplification (LAMP) assay for routine scoring of Wolbachia in mosquitoes from laboratory cultures and the field, applicable to any setting. We show that this assay is a rapid and robust method for highly sensitive and specific detection of wAlbB Wolbachia infection within Aedes aegypti under a variety of conditions. We test the quantitative nature of the assay by evaluating pooled mixtures of Wolbachia-infected and uninfected mosquitoes and show that it is capable of estimating infection frequencies, potentially circumventing the need to perform large-scale individual analysis for wAlbB infection status in the course of field monitoring. These results indicate that LAMP assays are useful for routine screening particularly under field conditions away from laboratory facilities.\n\nImportanceReleases of mosquitoes infected with strains of Wolbachia bacteria are expanding around the world because this bacterium that lives inside cells provides an effective tool to suppress mosquito populations and the ability of mosquitoes to transmit viruses. The success of the release programs relies on rapid and effective means of detecting Wolbachia and scoring their frequencies in mosquitoes for quality control and for assessing the success of releases. Here we test a \"LAMP\" (Loop-mediated isothermal amplification) assay for robust detection of Wolbachia infections in laboratory and field mosquito populations. We show that the assay can detect the bacteria when present at a low density in samples, and with a high degree of reproducibility. The assay uses a simple protocol which requires minimal training. It can readily detect Wolbachia in mosquitoes obtained from traps that are routinely used in field surveys. The assay should be cost effective in a variety of settings.

genetics↗

Does membrane feeding compromise the quality of Aedes aegypti mosquitoes?

Modified Aedes aegypti mosquitoes are being mass-reared for release in disease control programs around the world. Releases involving female mosquitoes rely on them being able to seek and feed on human hosts. To facilitate the mass-production of mosquitoes for releases, females are often provided blood through artificial membrane feeders. When reared across generations there is a risk that mosquitoes will adapt to feeding on membranes and lose their ability to feed on human hosts. To test adaptation to membrane feeding, we selected replicate populations of Ae. aegypti for feeding on either human arms or membrane feeders for at least 8 generations. Membrane-selected populations suffered fitness costs, likely due to inbreeding depression arising from bottlenecks. Membrane-selected females had higher feeding rates on membranes than human-selected ones, suggesting adaptation to membrane feeding, but they maintained their attraction to host cues and feeding ability on humans despite a lack of selection for these traits. Host-seeking ability in small laboratory cages did not differ between populations selected on the two blood sources, but membrane-selected females were compromised in a semi-field enclosure where host-seeking was tested over a longer distance. Our findings suggest that Ae. aegypti may adapt to feeding on blood provided artificially, but this will not substantially compromise field performance or affect experimental assessments of mosquito fitness. However, large population sizes during mass rearing with membrane feeders should be maintained to avoid bottlenecks which lead to inbreeding depression.\n\nAuthor summaryWith modified mosquitoes being mass-reared for release in disease control programs, there is interest in understanding factors that can affect the quality of release stocks. In the laboratory, membrane feeding devices are often used to provide blood to female mosquitoes which they need to lay eggs. Over time, mosquitoes could adapt to membrane feeding and lose their preference for (or ability to feed on) natural hosts. Adaptation could affect the performance of lab-reared mosquitoes when deployed in the field, especially if the success of disease control programs relies on female reproduction such as in gene drive or Wolbachia-based approaches. We tested to see if Aedes aegypti mosquitoes, the principal vectors of dengue virus, would adapt to feeding on blood provided via membranes. We found some evidence of adaptation, with membrane-selected mosquitoes having higher feeding rates on membranes, but this didnt affect their ability to feed on humans or their attraction to host cues. Although membrane feeding alone does not substantially affect mosquito quality, it can lead to inbreeding depression if populations pass through bottlenecks as they adapt to feeding on blood provided artificially. Our results may inform mass-rearing programs involving the release of modified female mosquitoes.

evolutionary biology↗

Measuring the host-seeking ability of Aedes aegypti destined for field release

Host-seeking is an essential process in mosquito reproduction. Field releases of modified mosquitoes for population transformation rely on successful host-seeking by female mosquitoes, but host-seeking ability is rarely tested in a realistic context. We tested the host-seeking ability of female Aedes aegypti mosquitoes using a semi-field system. Females with different Wolbachia infection types (wMel-, wAlbB-infected and uninfected) or from different origins (laboratory and field) were released at one end of a semi-field cage and recaptured as they landed on human experimenters fifteen meters away. Mosquitoes from each population were then identified with molecular tools or through marking with a consistent weight of fluorescent powder. Wolbachia-infected and uninfected populations had similar average durations to landing and overall recapture proportions, as did laboratory and field-sourced A. aegypti. These results suggest that the host-seeking ability of mosquitoes is not negatively affected by Wolbachia infection or long-term laboratory maintenance. This method provides an approach to study the host-seeking ability of mosquitoes across a long distance which will be useful when evaluating strains of mosquitoes that are planned for releases into the field to suppress arbovirus transmission. An adjustment of this method may also be useful in sterile insect release programs because male host-seeking and swarming around female feeding sites can also be investigated.

animal behavior and cognition↗

Effects of alternative blood sources on Wolbachia infected Aedes aegypti females within and across generations

Wolbachia bacteria have been identified as a tool for reducing the transmission of arboviruses transmitted by Aedes aegypti. Research groups around the world are now mass rearing Wolbachia infected Ae. aegypti for deliberate release. We investigated the fitness impact of a crucial element of mass rearing: the blood meal required by female Ae. aegypti to lay eggs. Although Ae. aegypti almost exclusively feed on human blood, it is often difficult to use human blood in disease-endemic settings. When females were fed on sheep or pig blood rather than human blood, egg hatch rates decreased in all three lines tested (uninfected, or infected by wMel, or wAlbB Wolbachia). This finding was particularly pronounced when fed on sheep blood, although fecundity was not affected. Some of these effects persisted after an additional generation on human blood. Attempts to keep populations on sheep and pig blood sources only partly succeeded, suggesting that strong adaptation is required to develop a stably infected line on an alternative blood source. There was a decrease in Wolbachia density when Wolbachia Ae. aegypti were fed on non-human blood sources, although density was higher again in lines kept for multiple generations on the alternate sources. These findings suggest that sheep and pig blood will entail a cost when used as alternative blood sources which needs to be taken into account when considering mass release.

evolutionary biology↗

Small females prefer small males: size assortative mating in Aedes aegypti mosquitoes

With Aedes aegypti mosquitoes now being released in field programs aimed at disease suppression, there is interest in identifying factors influencing the mating and invasion success of released mosquitoes. One factor that can increase release success is size: released males may benefit competitively from being larger than their field counterparts. However, there could be a risk in releasing only large males if small field females avoid these males and instead prefer small males. Here we investigate this risk by evaluating mating success for mosquitoes differing in size. We measured mating success indirectly by coupling size with Wolbachia-infected or uninfected mosquitoes and scoring cytoplasmic incompatibility as a way of estimating relative mating success. Large females showed no evidence of a mating preference, whereas small males were relatively more successful than large males when mating with small females, exhibiting an advantage of around 20-25%. Because field females typically encompass a wide range of sizes while laboratory reared (and released) males typically fall into a narrow size range of large mosquitoes, these patterns can influence the success of release programs which rely on cytoplasmic incompatibility to suppress populations and initiate replacement invasions. Releases could include some small males generated under low food or crowded conditions to counter this issue, although this would need to be weighed against issues associated with costs of producing males of various size classes.

animal behavior and cognition↗

A comprehensive assessment of inbreeding and laboratory adaptation in Aedes aegypti mosquitoes

Modified Aedes aegypti mosquitoes reared in laboratories are being released around the world to control wild mosquito populations and the diseases they transmit. Several efforts have failed due to poor competitiveness of the released mosquitoes. We hypothesized that colonized mosquito populations could suffer from inbreeding depression and adapt to laboratory conditions, reducing their performance in the field. We established replicate populations of Ae. aegypti mosquitoes collected from Queensland, Australia, and maintained them in the laboratory for twelve generations at different census sizes. Mosquito colonies maintained at small census sizes ([≤]100 individuals) suffered from inbreeding depression due to low effective population sizes which were only 25% of the census size as estimated by SNP markers. Populations that underwent full-sib mating for 9 consecutive generations had greatly reduced performance across all traits measured. We compared the established laboratory populations with their ancestral population resurrected from quiescent eggs for evidence of laboratory adaptation. The overall performance of laboratory populations maintained at a large census size (400 individuals) increased, potentially reflecting adaptation to artificial rearing conditions. However most individual traits were unaffected, and patterns of adaptation were not consistent across populations. Differences between replicate populations may indicate that founder effects and drift affect experimental outcomes. Though we find limited evidence of laboratory adaptation, mosquitoes maintained at low population sizes can clearly suffer fitness costs, compromising the success of \"rear and release\" strategies for arbovirus control.

evolutionary biology↗