One-Pot Visual Detection of African Swine Fever Virus Using CRISPR-Cas12a

African swine fever virus (ASFV) is a leading cause of worldwide agricultural loss. ASFV is a highly contagious and lethal disease for both domestic and wild pigs, which has brought enormous economic losses to a number of countries. Conventional methods, such as general polymerase chain reaction and isothermal amplification, are time-consuming, instrument-dependent, and unsatisfactorily accurate. Therefore, rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we created a one-pot visual detection system for ASFV with CRISPR/Cas12a technology combined with LAMP or RPA. A mineral oil sealing strategy was adopted to mitigate sample cross-contamination between parallel vials during high-throughput testing. Furthermore, the blue fluorescence signal produced by ssDNA reporter could be observed by the naked eye without any dedicated instrument. For CRISPR-RPA system, detection could be completed within 40 min with advantageous sensitivity. While CRISPR-LAMP system could complete it within 60 min with a high sensitivity of 5.8 × 102 copies/μl. Furthermore, we verified such detection platforms display no cross-reactivity with other porcine DNA or RNA viruses. Both CRISPR-RPA and CRISPR-LAMP systems permit highly rapid, sensitive, specific, and low-cost Cas12a-mediated visual diagnostic of ASFV for point-of-care testing (POCT) applications

Speciation and the evolution of male breeding coloration in darters

The means by which sexual selection can promote speciation is a topic of much debate and study. Darters, a species-rich clade of freshwater fishes that exhibit widespread sexual dichromatism, may represent a prime example of speciation by sexual selection. I investigated mechanisms of sexual selection and reproductive isolation in two closely related, ecologically and behaviorally similar species: the rainbow darter (Etheostoma caeruleum) and the orangethroat darter (E. spectabile). First, I quantified variation in male breeding coloration across multiple populations of E. caeruleum and E. spectabile using digital photography and image processing software. Male color traits in both species can be categorized as “blue” and “red” components, which are based on chromoprotein and carotenoid pigments, respectively. Blue colors tended to be more conserved and better correlated with body size, whereas red colors tended to be more diverse among species and populations and less correlated with body size. These disparate patterns suggest that the blue and red components of male coloration serve different functions and/or are subject to different selective forces. I then investigated the specifics of behavioral isolation between E. caeruleum and E. spectabile, and whether species discrimination is mediated by male coloration. I observed interactions between male E. spectabile, female E. spectabile, and male E. caeruleum under different lighting conditions that permitted or impaired the fish’s perception of red coloration. Species recognition was controlled by males, who preferentially pursued conspecific females and directly aggression toward conspecific males. Females did not exhibit overt preference for any type of male, instead responding proportionately to the amount of male pursuit. Disrupting color perception inhibited male-male aggression, suggesting that male coloration allows males to identify conspecific rivals in the context of male-male competition. As previous studies have found no effect of female choice on male fitness in E. caeruleum or E. spectabile, I tested whether male-male competition underlies sexual selection. I allowed multiple male E. spectabile to compete for a single female and looked for phenotypic correlates of reproductive success. Relatively larger males were competitively superior and initiated more spawning events with females. After correcting for size, I also found that the relative quality and quantity of male coloration was correlated with success in guarding the female against rivals and in spawning with the female. These results indicate that male coloration is under sexual selection through male-male competition. Apart from sexual selection, behavioral isolation may also evolve through natural selection against heterospecific mating, i.e. reinforcement. I performed conspecific and heterospecific crosses using E. caeruleum and E. spectabile and compared reproductive isolation between these species in a sympatric population versus allopatric populations. Reproductive isolation was high between female E. caeruleum and male E. spectabile in both sympatry and allopatry. In contrast, reproduction isolation was high between female E. spectabile and male E. caeruleum in sympatry but low in allopatry. This pattern is consistent with reinforcement acting asymmetrically on behavioral isolation between these species. Finally, I conducted a long-term investigation of postzygotic isolation between E. caeruleum and E. spectabile. I created purebred and hybrid F1 fish via artificial fertilization and raised them under common garden conditions. Fertilization success, hatching success, and larval survival to 10 months did not differ between purebred and hybrid families, indicating little hybrid inviability. However, hybrid families from both reciprocal heterospecific crosses were heavily biased toward males, while the purebred families did not differ from the 1:1 sex ratio found in nature. This sex ratio distortion suggests that heterospecific spawning between E. caeruleum and E. spectabile is detrimental, and provides a possible mechanism for driving reinforcement between these species

zhou&fuller_malecomp

Behavioral and chromatic data for male E. spectabil

color_behavior_data

The first sheet in this file includes color pattern data for male orangethroat (OT) and rainbow (RB) darters and for male wild-caught hybrids (WH) and lab-raised hybrids (LH). Df = dorsal fin, Af = anal fin, Lat = lateral side, R = red, B = blue, PR = proportion red, PB = proportion blue. The second sheet in this file includes data from the wild-caught hybrid male aggression trials. The third sheet in this file includes data from the wild-caught hybrid male dichotomous mate choice trials

Data from: Hybridization and postzygotic isolation promote reinforcement of male mating preferences in a diverse group of fishes with traditional sex roles

Behavioral isolation is thought to arise early in speciation due to differential sexual and/or natural selection favoring different preferences and traits in different lineages. Alternatively, behavioral isolation can arise due to reinforcement favoring traits and preferences that prevent maladaptive hybridization. In darters, female preference for male coloration has been hypothesized to drive speciation, because behavioral isolation evolves before F1 inviability. However, as with many long-lived organisms, the fitness of second generation hybrids has not been assessed because raising animals to adulthood in the lab is challenging. Recently, reinforcement of male preferences has been implicated in darters because male preference for conspecific females is high in sympatry but absent in allopatry in multiple species pairs. The hypothesis that reinforcement accounts for behavioral isolation in sympatry assumes that hybridization and postzygotic isolation are present. Here, we used genomic and morphological data to demonstrate that hybridization is ongoing between orangethroat and rainbow darters and used hybrids collected from nature to measure postzygotic barriers across two hybrid generations. We observed sex ratio distortion in adult F1s and a dramatic reduction in backcross survival. Our findings indicate that selection to avoid hybridization promotes the evolution of male-driven behavioral isolation via reinforcement in this system

Data from: The effects of age, sex, and habitat on body size and shape of the blackstripe topminnow, Fundulus notatus

Lake and stream habitats pose a variety of challenges to fishes due to differences in variables such as water velocity, habitat structure, prey community, and predator community. These differences can cause divergent selection on body size and/or shape. Here, we measured sex, age, length, and eight different morphological traits of the blackstripe topminnow, Fundulus notatus, from 19 lake and stream populations across four river drainages in central Illinois. Our goal was to determine whether size and shape differed consistently between lake and stream habitats across drainages. We also considered the effects of age and sex as they may affect size and morphology. We found large differences in body size of age 1 topminnows where stream fish were generally larger than lake fish. Body shape mainly varied as a function of sex. Adult male topminnows had larger morphological traits (with the exception of body width) than females, in particular longer dorsal and anal base lengths. Subtle effects of habitat were present. Stream fish had a longer dorsal fin base than lake fish. These phenotypic patterns may be the result of genetic and/or environmental variation. As these lakes are human-made, the observed differences, if genetic, would have had to occur relatively rapidly (within about 100 years)

Fundulus notatus raw data

Excel file containing the raw data (morphometric measurements as well as data on population, drainage, habitat, age, and sex)