Effects of Functional Depletion of Doublesex on Male Development in the Sawfly, Athalia rosae

The doublesex (dsx) gene, which encodes a transcription factor, regulates sexual differentiation in insects. Sex-specific splicing of dsx occurs to yield male- and female-specific isoforms, which promote male and female development, respectively. Thus, functional disruption of dsx leads to an intersexual phenotype in both sexes. We previously identified a dsx ortholog in the sawfly, Athalia rosae. Similar to dsx in other insects, dsx in the sawfly yields different isoforms in males and females as a result of alternative splicing. The sawfly exploits a haplodiploid mode of reproduction, in which fertilized eggs develop into diploid females, whereas unfertilized eggs parthenogenetically develop into haploid males. In the present study, we knocked down the A. rosae ortholog of dsx (Ardsx) during several developmental stages with repeated double-stranded RNA (dsRNA) injections. Knockdown of Ardsx via parental RNA interference (RNAi), which enables knockdown of genes in offspring embryos, led to a lack of internal and external genitalia in haploid male progeny. Additional injection of dsRNA targeting Ardsx in these animals caused almost complete male-to-female sex reversal, but the resulting eggs were infertile. Notably, the same knockdown approach using diploid males obtained by sib-crossing caused complete male-to-female sex reversal; they were morphologically and behaviorally females. The same RNAi treatment did not affect female differentiation. These results indicate that dsx in the sawfly is essential for male development and its depletion caused complete male-to-female sex reversal. This is the first demonstration of functional depletion of dsx not causing intersexuality but inducing total sex reversal in males instead.

Induced triploidy reduces mercury bioaccumulation in a piscivorous fish

We compared mercury bioaccumulation in triploid and diploid walleye (Stizostedion vitreum) in Narraguinnep Reservoir, Colorado, USA, and made several hypotheses that sex- and ploidy-specific differences in the allocation of energy towards reproductive development would affect mercury bioaccumulation. We tested our hypotheses with linear regression and a bioenergetics model informed by field data. We found diploid walleye had 28-31% higher mercury concentrations on average than triploids, but there were no differences between sexes of the same ploidy. Triploids of mature age exhibited minimal gonadal development when compared to diploids. After accounting for reproductive investment, the bioenergetics model accounted for most of the observed difference in average mercury concentration between ploidies for females. Conversely, the energetic cost of producing testes was low, and gonadal development could not explain observed patterns for males. Costs associated with elevated swimming activity and metabolism by diploid males relative to other groups could explain the difference but requires further investigation. The use of triploid fish in stocking programs could prove useful for reducing mercury in fish destined for human consumption.

Gynogenetic diploids, tetraploids, or octoploids, and a path to polyploidy in Anuran Amphibians

Unreduced gametes have been implicated in the evolution of polyploid species of plants and animals and are normally produced by female anuran amphibians. Such eggs may initiate the evolution of polyploid species that have independently arisen in several anuran families. Polyploid females could also produce unreduced eggs that might lead to species with higher ploidy levels or their eggs may develop gynogenetically to reduce the ploidy level. Diploid Hyla chrysoscelis (2n=24) and tetraploid H. versicolor (4n=48) are sibling cryptic species of North American Grey Treefrogs. Artificial crosses using H. versicolor females and genetically distant diploid males were performed to produce haploid H. versicolor and to assess the production of unreduced eggs in this tetraploid species. Gynogenetic diploid (haploid H. versicolor), allotriploid, gynogenetic tetraploid, allopentaploid, autohexaploid, and gynogenetic octoploid tadpoles were confirmed using chromosome counts from tadpole tail tip squashes. Transformation and survival of the different ploidies varied. Gynogenetic diploids transformed but expressed aspects of the haploid syndrome and died before or shortly after transformation.

Origin, behaviour, and genetics of reproductive workers in an invasive ant

Background Worker reproduction has an important influence on the social cohesion and efficiency of social insect colonies, but its role in the success of invasive ants has been neglected. We used observations of 233 captive colonies, laboratory experiments, and genetic analyses to investigate the conditions for worker reproduction in the invasive Anoplolepis gracilipes (yellow crazy ant) and its potential cost on interspecific defence. We determined the prevalence of worker production of males and whether it is triggered by queen absence; whether physogastric workers with enlarged abdomens are more likely to be reproductive, how normal workers and physogastric workers compare in their contributions to foraging and defence; and whether worker-produced males and males that could have been queen- or worker-produced differ in their size and heterozygosity. Results Sixty-six of our 233 captive colonies produced males, and in 25 of these, some males could only have been produced by workers. Colonies with more workers were more likely to produce males, especially for queenless colonies. The average number of days between the first appearance of eggs and adult males in our colonies was 54.1 ± 10.2 (mean ± SD, n = 20). In our laboratory experiment, queen removal triggered an increase in the proportion of physogastric workers. Physogastric workers were more likely to have yolky oocytes (37–54.9%) than normal workers (2–25.6%), which is an indicator of fertile or trophic egg production. Physogastric workers were less aggressive during interspecific aggression tests and foraged less than normal workers. The head width and wing length of worker-produced males were on average 4.0 and 4.3% greater respectively than those of males of undetermined source. Our microsatellite DNA analyses indicate that 5.5% of worker-produced males and 14.3% of males of undetermined source were heterozygous, which suggests the presence of diploid males and/or genetic mosaics in A. gracilipes. Conclusions Our experimental work provides crucial information on worker reproduction in A. gracilipes and its potential cost to colony defence. The ability of A. gracilipes workers to produce males in the absence of queens may also contribute to its success as an invasive species if intranidal mating can take place between virgin queens and worker-produced males.

Origin, behaviour, and genetics of reproductive workers in an invasive ant

Background: Worker reproduction has an important influence on the social cohesion and efficiency of social insect colonies, but its role in the success of invasive ants has been neglected. We used observations of 233 captive colonies, laboratory experiments, and genetic analyses to investigate the conditions for worker reproduction in the invasive Anoplolepis gracilipes (yellow crazy ant) and its potential cost on interspecific defence. We determined the prevalence of worker production of males and whether it is triggered by queen absence; whether physogastric workers with enlarged abdomens are more likely to be reproductive, how normal workers and physogastric workers compare in their contributions to foraging and defence; and whether worker-produced males and males that could have been queen- or worker-produced differ in their size and heterozygosity.Results: Sixty-six of our 233 captive colonies produced males, and in 25 of these, some males could only have been produced by workers. Colonies with more workers were more likely to produce males, especially for queenless colonies. The average number of days between the first appearance of eggs and adult males in our colonies was 54.1± 10.2 (mean ± SD, n=20). In our laboratory experiment, queen removal triggered an increase in the proportion of physogastric workers. Physogastric workers were more likely to have yolky oocytes (37-54.9%) than normal workers (2-25.6%), which is an indicator of fertile or trophic egg production. Physogastric workers were less aggressive during interspecific aggression tests and foraged less than normal workers. The head width and wing length of worker-produced males were on average 4.0% and 4.3% greater respectively than those of males of undetermined source. Our microsatellite DNA analyses indicate that 5.5% of worker-produced males and 14.3% of males of undetermined source were heterozygous, which suggests the presence of diploid males and/or genetic mosaics in A. gracilipes. Conclusions: Our experimental work provides crucial information on worker reproduction in A. gracilipes and its potential cost to colony defence. The ability of A. gracilipes workers to produce males in the absence of queens may also contribute to its success as an invasive species if intranidal mating can take place between virgin queens and worker-produced males.

No severe genetic bottleneck in a rapidly range-expanding bumblebee pollinator

Genetic bottlenecks can limit the success of populations colonizing new ranges. However, successful colonizations can occur despite bottlenecks, a phenomenon known as the genetic paradox of invasion. Eusocial Hymenoptera such as bumblebees (Bombusspp.) should be particularly vulnerable to genetic bottlenecks, since homozygosity at the sex-determining locus leads to costly diploid male production (DMP). The Tree Bumblebee (Bombus hypnorum) has rapidly colonized the UK since 2001 and has been highlighted as exemplifying the genetic paradox of invasion. Using microsatellite genotyping, combined with the first genetic estimates of DMP in UKB. hypnorum, we tested two alternative genetic hypotheses (‘bottleneck’ and ‘gene flow’ hypotheses) forB. hypnorum's colonization of the UK. We found that the UK population has not undergone a recent severe genetic bottleneck and exhibits levels of genetic diversity falling between those of widespread and range-restrictedBombusspecies. Diploid males occurred in 15.4% of reared colonies, leading to an estimate of 21.5 alleles at the sex-determining locus. Overall, the findings show that this population is not bottlenecked, instead suggesting that it is experiencing continued gene flow from the continental European source population with only moderate loss of genetic diversity, and does not exemplify the genetic paradox of invasion.

Origin, Behaviour, and Genetics of Reproductive Workers in an Invasive Ant

Background: Worker reproduction has an important influence on the social cohesion and efficiency of social insect colonies, but its role in the success of invasive ants has been neglected. We used observations of 233 captive colonies, laboratory experiments, and genetic analyses to investigate the conditions for worker reproduction in the invasive Anoplolepis gracilipes (yellow crazy ant) and its potential cost on interspecific defence. We determined how common worker production of males is and whether it is triggered by queen absence; whether physogastric workers with enlarged abdomens are more likely to be reproductive, how normal workers and physogastric workers compare in their contributions to colony labour and defence; and whether worker-produced males and males that could have been queen- or worker-produced differ in their size and heterozygosity. Results: Sixty-six of our 233 captive colonies produced males, and in 25 of these, some males could only have been produced by workers. Colonies with more workers were more likely to produce males, especially for queenless colonies. The average number of days between the first appearance of eggs and adult males in our colonies was 54.1± 10.2 (mean ± SD, n=20). In our laboratory experiment, queen removal triggered an increase in the proportion of physogastric workers. Physogastric workers were more likely to have yolky oocytes (37-54.9%) than normal workers (2-25.6%), which is an indicator of fertile or trophic egg production. Physogastric workers were less aggressive during interspecific aggression tests and foraged less than normal workers. The head width and wing length of worker-produced males were on average 4.0% and 4.3% greater respectively than those of males of undetermined source. Our microsatellite DNA analyses indicate that 4.9% of worker-produced males and 21.1% of males of undetermined source were heterozygous, which suggests the presence of diploid males and/or genetic mosaics in A. gracilipes. Conclusions: Our experimental work provides crucial information on worker reproduction in A. gracilipes and its potential cost to colony defence. The ability of A. gracilipes workers to produce males in the absence of queens may also contribute to its success as an invasive species if intranidal mating can take place between virgin queens and worker-produced males.

Managing incursions of Vespa velutina nigrithorax in the UK: an emerging threat to apiculture

Vespa velutina nigrithorax is an invasive species of hornet accidentally introduced into Europe in 2004. It feeds on invertebrates, including honey bees, and represents a threat to European apiculture. In 2016, the first nest of this hornet was detected and destroyed on mainland UK. A further 8 nests were discovered between 2016 and 2019. Nest dissection was performed on all nests together with microsatellite analyses of different life stages found in the nests to address the reproductive output and success of nests found in the UK. None of the nests had produced the next generation of queens. Follow-up monitoring in those regions detected no new nests in the following years. Diploid males were found in many UK nests, while microsatellite analysis showed that nests had low genetic diversity and the majority of queens had mated with one or two males. All UK nests derived from the European zone of secondary colonisation, rather than from the native range of the species. None of the nests discovered so far have been direct offspring of another UK nest. The evidence suggests that these nests were separate incursions from a continental population rather than belonging to a single established UK population of this pest.