In this mini-review, we provide an up-to-date overview of the delivery methods that have been used for CRISPR/Cas9 genomic editing in crustacean species. With embryonic microinjection as the main workforce for delivering CRISPR/Cas9 reagents, biologists working with crustacean species have to tackle the technical challenges involved in microinjection. We use examples of three crustacean species (the branchiopod Daphnia, amphipod Parhyale hawaiensis, and decapod Exopalaemon carinicauda) to provide a technical guide for embryonic microinjection. Moreover, we outline two potentially useful new techniques for delivering CRISPR/Cas9 components into crustaceans, i.e., Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and electroporation.

Theories predict that directional selection during adaptation to a novel habitat results in elevated meiotic recombination rate. Yet the lack of population-level recombination rate data leaves this hypothesis untested in natural populations. Here we examine the population-level recombination rate variation in two incipient ecological species, the microcrustacean Daphnia pulex (an ephemeral-pond species) and D. pulicaria (a permanent-lake species). The divergence of D. pulicaria from D. pulex involved habitat shifts from pond to lake habitats as well as strong local adaptation due to directional selection. Using a novel single-sperm genotyping approach, we estimated the male-specific recombination rate of two linkage groups in multiple populations of each species in common garden experiments and identified a significantly elevated recombination rate in D. pulicaria. Most importantly, population genetic analyses show that the divergence in recombination rate between these two species is most likely due to divergent selection in distinct ecological habitats rather than neutral evolution.

Mutation rate in the nuclear genome differs between sexes, with males contributing more mutations than females to their offspring. The male-biased mutation rates in the nuclear genome is most likely to be driven by a higher number of cell divisions in spermatogenesis than in oogenesis, generating more opportunities for DNA replication errors. However, it remains unknown whether male-biased mutation rates are present in mitochondrial DNA (mtDNA). Although mtDNA is maternally inherited and male mtDNA mutation typically does not contribute to genetic variation in offspring, male mtDNA mutations are critical for male reproductive health. In this study, we measured male mtDNA mutation rate using publicly available whole-genome sequences of single sperm of the freshwater microcrustacean Using a stringent mutation detection pipeline, we found that the male mtDNA mutation rate is 3.32 × 10 per site per generation. All the detected mutations are heteroplasmic base substitutions, with 57% of mutations converting G/C to A/T nucleotides. Consistent with the male-biased mutation in the nuclear genome, the male mtDNA mutation rate in is approximately 20 times higher than the female rate per generation. We propose that the elevated mutation rate per generation in male mtDNA is consistent with an increased number of cell divisions during male gametogenesis.

Theories predict that directional selection during adaptation to a novel habitat results in elevated meiotic recombination rate. Yet the lack of population-level recombination rate data leaves this hypothesis untested in natural populations. Here we examine the population-level recombination rate variation in two incipient ecological species, the microcrustacean Daphnia pulex (an ephemeral-pond species) and D. pulicaria (a permanent-lake species). The divergence of D. pulicaria from D. pulex involved habitat shifts from pond to lake habitats as well as strong local adaptation due to directional selection. Using a novel single-sperm genotyping approach, we estimated the male-specific recombination rate of two linkage groups in multiple populations of each species in common garden experiments and identified a significantly elevated recombination rate in D. pulicaria. Most importantly, population genetic analyses show that the divergence in recombination rate between these two species is most likely due to divergent selection in distinct ecological habitats rather than neutral evolution.Significance statementWhether directional selection during adaptation to a novel habitat results in elevated meiotic recombination remains largely untested in natural populations. This work examines the population-level recombination rate in two closely related microcructacean species Daphnia pulex and D. pulicaria using single-sperm genotyping approach. Recombination rate data from two linkage groups show elevated recombination rates in D. pulicaria whose divergence from D. pulex is accompanied by a habitat shift. Importantly, population genetic analysis suggests that this divergence of recombination is likely adaptive rather than neutral.

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