A comprehensive framework for the delimitation of species within the Bemisia tabaci cryptic complex, a global pest-species group.

Identifying cryptic species poses a substantial challenge to both biologists and naturalists due to morphological similarities. Bemisia tabaci is a cryptic species complex containing more than 44 putative species; several of which are currently among the world's most destructive crop pests. Interpreting and delimiting the evolution of this species complex has proved problematic. To develop a comprehensive framework for species delimitation and identification, we evaluated the performance of distinct data sources both individually and in combination among numerous samples of the B. tabaci species complex acquired worldwide. Distinct datasets include full mitogenomes, single-copy nuclear genes, restriction site-associated DNA sequencing, geographic range, host speciation, and reproductive compatibility datasets. Phylogenetically, our well-supported topologies generated from three dense molecular markers highlighted the evolutionary divergence of species of the B. tabaci complex and suggested that the nuclear markers serve as a more accurate representation of B. tabaci species diversity. Reproductive compatibility datasets facilitated the identification of at least 17 different cryptic species within our samples. Native geographic range information provides a complementary assessment of species recognition, while the host range datasets provide low rate of delimiting resolution. We further summarized different data performances in species classification when compared with reproductive compatibility, indicating that combination of mtCOI divergence, nuclear markers, geographic range provide a complementary assessment of species recognition. Finally, we represent a model for understanding and untangling the cryptic species complexes based on the evidence from this study and previously published articles.

Draft genome assemblies of the avian louse Brueelia nebulosa and its associates using long-read sequencing from an individual specimen.

Sequencing high molecular weight (HMW) DNA with long-read and linked-read technologies has promoted a major increase in more complete genome sequences for nonmodel organisms. Sequencing approaches that rely on HMW DNA have been limited to larger organisms or pools of multiple individuals, but recent advances have allowed for sequencing from individuals of small-bodied organisms. Here, we use HMW DNA sequencing with PacBio long reads and TELL-Seq linked reads to assemble and annotate the genome from a single individual feather louse (Brueelia nebulosa) from a European Starling (Sturnus vulgaris). We assembled a genome with a relatively high scaffold N50 (637 kb) and with BUSCO scores (96.1%) comparable to louse genomes assembled from pooled individuals. We annotated a number of genes (10,938) similar to the human louse (Pediculus humanus) genome. Additionally, calling phased variants revealed that the Brueelia genome is more heterozygous (∼1%) then expected for a highly obligate and dispersal-limited parasite. We also assembled and annotated the mitochondrial genome and primary endosymbiont (Sodalis) genome from the individual louse, which showed evidence for heteroplasmy in the mitogenome and a reduced genome size in the endosymbiont compared to its free-living relative. Our study is a valuable demonstration of the capability to obtain high-quality genomes from individual small, nonmodel organisms. Applying this approach to other organisms could greatly increase our understanding of the diversity and evolution of individual genomes.

Independent evolution of highly variable, fragmented mitogenomes of parasitic lice.

The mitochondrial genomes (mitogenomes) of bilaterian animals are highly conserved structures that usually consist of a single circular chromosome. However, several species of parasitic lice (Insecta: Phthiraptera) possess fragmented mitogenomes, where the mitochondrial genes are present on separate, circular chromosomes. Nevertheless, the extent, causes, and consequences of this structural variation remain poorly understood. Here, we combined new and existing data to better understand the evolution of mitogenome fragmentation in major groups of parasitic lice. We found strong evidence that fragmented mitogenomes evolved many times within parasitic lice and that the level of fragmentation is highly variable, including examples of heteroplasmic arrangements. We also found a significant association between mitochondrial fragmentation and signatures of relaxed selection. Mitochondrial fragmentation was also associated with changes to a lower AT%, possibly due to differences in mutation biases. Together, our results provide a significant advance in understanding the process of mitogenome fragmentation and provide an important perspective on mitochondrial evolution in eukaryotes.

Gene arrangement, phylogeny and divergence time estimation of mitogenomes in Thrips.

BACKGROUND: The metazoan mitogenomes usually display conserved gene arrangement while thrips are known for their extensive gene rearrangement, and duplication of the control region. METHODS AND RESULT: We sequenced complete mitogenomes of eight species of thrips to determine the gene arrangement, phylogeny and divergence time estimation. All contain 37 genes and one control region, (CR) except four species with two CRs. Duplicated tRNAs were detected in Mycterothrips nilgiriensis and Thrips florum. nad4-nad4L were not found adjacent to each other in Phibalothrips peringueyi and Plicothrips apicalis. Both Bayesian and likelihood phylogenetic analyses of thrips mitogenomes supported the monophyly of two suborders (Terebrantia and Tubulifera) and the two largest families (Phlaeothripidae and Thripidae). Out of seven earlier proposed ancestral gene blocks, six are conserved in Panchaetothripinae, three in Thripinae and two in Phlaeothripidae. Additionally, eight Thrips Gene Blocks were identified, of which, three conserved in Tubulifera, four in Terebrantia, and one only in Aeolothripidae. Forty-two gene boundaries (15 from previous study + 27 new) were identified. The molecular divergence time is estimated for the order Thysanoptera and suggested that these insects may have been diversified from hemipterans in the late Permian period. The most recent ancestors belong to family Thripidae and Phlaeothripidae, which were diversified in upper Cretaceous period and showed higher rates of rearrangement from the ancestral gene order. CONCLUSIONS: The current study is the first largest effort to provide the new insights into the mitogenomic features, gene arrangement, phylogeny and divergence time estimation of thrips belonging to the order Thysanoptera.

How are the mitochondrial genomes reorganized in Hexapoda? Differential evolution and the first report of convergences within Hexapoda.

The evolution of the Hexapoda mitochondrial genome has been the focus of several genetic and evolutionary studies over the last decades. However, they have concentrated on certain taxonomic orders of economic or health importance. The recent increase of mitochondrial genomes sequencing of diverse taxonomic orders generates an important opportunity to clarify the evolution of this group of organisms. However, there is no comparative study that investigates the evolution of the Hexapoda mitochondrial genome. In order to verify the level of rearrangement and the mitochondrial genome evolution, we performed a comparative genomic analysis of the Hexapoda mitochondrial genome available in the NCBI database. Using a combination of bioinformatics methods to carefully examine the mitochondrial gene rearrangements in 1198 Hexapoda species belonging to 32 taxonomic orders, we determined that there is a great variation in the rate of rearrangement by gene and by taxonomic order. A higher rate of genetic reassortment is observed in Phthiraptera, Thysanoptera, Protura, and Hymenoptera; compared to other taxonomic orders. Twenty-four events of convergence in the genetic order between different taxonomic orders were determined, most of them not previously reported; which proves the great evolutionary dynamics within Hexapoda.

Polyzosteria cockroaches in Tasmania (Blattodea: Blattidae: Polyzosteriinae) represent a new, endemic species, with allopatric alpine and coastal sub-populations.

We describe the endemic Tasmanian cockroach, Polyzosteria yingina sp. nov. (Henry), 78 years after it was first documented. Evidence from morphology, biogeography and CO1 barcodes is used to distinguish this species from related mainland Australian taxa it has previously been confused with. Polyzosteria yingina sp. nov. has two strongly allopatric populations: a compact alpine population above 1000m and a dispersed east coastal one at sealevel. However, mitochondrial Control Region D-loop molecular analysis suggests a single species identity for these disparate populations. Detailed internal and external morphological descriptions and photographs of living and preserved type material are presented. We also speculate on some hypotheses which could account for the unusual distribution of this charismatic insect.

Structure, gene order, and nucleotide composition of mitochondrial genomes in parasitic lice from Amblycera.

Parasitic lice have unique mitochondrial (mt) genomes characterized by rearranged gene orders, variable genome structures, and less AT content compared to most other insects. However, relatively little is known about the mt genomes of Amblycera, the suborder sister to all other parasitic lice. Comparing among nine different genera (including representative of all seven families), we show that Amblycera have variable and highly rearranged mt genomes. Some genera have fragmented genomes that vary considerably in length, whereas others have a single mt chromosome. Notably, these genomes are more AT-biased than most other lice. We also recover genus-level phylogenetic relationships among Amblycera that are consistent with those reported from large nuclear datasets, indicating that mt sequences are reliable for reconstructing evolutionary relationships in Amblycera. However, gene order data cannot reliably recover these same relationships. Overall, our results suggest that the mt genomes of lice, already know to be distinctive, are even more variable than previously thought.

Mitochondrial genomes of Columbicola feather lice are highly fragmented, indicating repeated evolution of minicircle-type genomes in parasitic lice.

Most animals have a conserved mitochondrial genome structure composed of a single chromosome. However, some organisms have their mitochondrial genes separated on several smaller circular or linear chromosomes. Highly fragmented circular chromosomes ("minicircles") are especially prevalent in parasitic lice (Insecta: Phthiraptera), with 16 species known to have between nine and 20 mitochondrial minicircles per genome. All of these species belong to the same clade (mammalian lice), suggesting a single origin of drastic fragmentation. Nevertheless, other work indicates a lesser degree of fragmentation (2-3 chromosomes/genome) is present in some avian feather lice (Ischnocera: Philopteridae). In this study, we tested for minicircles in four species of the feather louse genus Columbicola (Philopteridae). Using whole genome shotgun sequence data, we applied three different bioinformatic approaches for assembling the Columbicola mitochondrial genome. We further confirmed these approaches by assembling the mitochondrial genome of Pediculus humanus from shotgun sequencing reads, a species known to have minicircles. Columbicola spp. genomes are highly fragmented into 15-17 minicircles between ∼1,100 and ∼3,100 bp in length, with 1-4 genes per minicircle. Subsequent annotation of the minicircles indicated that tRNA arrangements of minicircles varied substantially between species. These mitochondrial minicircles for species of Columbicola represent the first feather lice (Philopteridae) for which minicircles have been found in a full mitochondrial genome assembly. Combined with recent phylogenetic studies of parasitic lice, our results provide strong evidence that highly fragmented mitochondrial genomes, which are otherwise rare across the Tree of Life, evolved multiple times within parasitic lice.

Quantifying Induced Polarization of Conductive Inclusions in Porous Media and Implications for Geophysical Measurements.

Induced polarization (IP) mapping has gained increasing attention in the past decades, as electrical induced polarization has been shown to provide interesting signatures for detecting the presence of geological materials such as clay, ore, pyrite, and potentially, hydrocarbons. However, efforts to relate complex conductivities associated with IP to intrinsic physical properties of the corresponding materials have been largely empirical. Here we present a quantitative interpretation of induced polarization signatures from brine-filled rock formations with conductive inclusions and show that new opportunities in geophysical exploration and characterization could arise. Initially tested with model systems with solid conductive inclusions, this theory is then extended and experimentally tested with nanoporous conductors that are shown to have a distinctive spectral IP response. Several of the tests were conducted with nano-porous sulfides (pyrite) produced by sulfate-reducing bacteria grown in the lab in the presence of a hydrocarbon source, as well as with field samples from sapropel formations. Our discoveries and fundamental understanding of the electrode polarization mechanism with solid and porous conductive inclusions suggest a rigorous new approach in geophysical exploration for mineral deposits. Moreover, we show how induced polarization of biologically generated mineral deposits can yield a new paradigm for basin scale hydrocarbon exploration.

A newly recorded Rickettsia of the Torix group is a recent intruder and an endosymbiont in the whitefly Bemisia tabaci.

The bacterium Rickettsia is found widely in phytophagous insects and often exerts profound effects on the phenotype and fitness of its hosts. Here, we decrypt a new, independent, phylogenetically ancient Torix Rickettsia endosymbiont found constantly in a laboratory line of an economically important insect Asia II 7, a putative species of the Bemisia tabaci whitefly complex (Hemiptera: Aleyrodidae), and occasionally in field whitefly populations. This new Rickettsia distributes throughout the body of its whitefly host. Genetically, compared to Rickettsia_bellii_MEAM1 found earlier in whiteflies, the new Rickettsia species has more gene families and pathways, which may be important factors in shaping specific symbiotic relationships. We propose the name 'Candidatus Rickettsia_Torix_Bemisia_tabaci (RiTBt)' for this new endosymbiont associated with whiteflies. Comparative genomic analyses indicate that RiTBi may be a relatively recent intruder in whiteflies given its low abundance in the field and relatively larger genome compared to Rickettsia_bellii_MEAM1.

Rearrangement and evolution of mitochondrial genomes in Thysanoptera (Insecta).

Prior to this study, complete mitochondrial genomes from Order Thysanoptera were restricted to a single family, the Thripidae, resulting in a biased view of their evolution. Here we present the sequences for the mitochondrial genomes of four additional thrips species, adding three extra families and an additional subfamily, thus greatly improving taxonomic coverage. Thrips mitochondrial genomes are marked by high rates of gene rearrangement, duplications of the control region and tRNA mutations. Derived features of mitochondrial tRNAs in thrips include gene duplications, anticodon mutations, loss of secondary structures and high gene translocation rates. Duplicated control regions are found in the Aeolothripidae and the 'core' Thripinae clade but do not appear to promote gene rearrangement as previously proposed. Phylogenetic analysis of thrips mitochondrial sequence data supports the monophyly of two suborders, a sister-group relationship between Stenurothripidae and Thripidae, and suggests a novel set of relationships between thripid genera. Ancestral state reconstructions indicate that genome rearrangements are common, with just eight gene blocks conserved between any thrips species and the ancestral insect mitochondrial genome. Conversely, 71 derived rearrangements are shared between at least two species, and 24 of these are unambiguous synapomorphies for clades identified by phylogenetic analysis. While the reconstructed sequence of genome rearrangements among the protein-coding and ribosomal RNA genes could be inferred across the phylogeny, direct inference of phylogeny from rearrangement data in MLGO resulted in a highly discordant set of relationships inconsistent with both sequence-based phylogenies and previous morphological analysis. Given the demonstrated rates of genomic evolution within thrips, extensive sampling is needed to fully understand these phenomena across the order.

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini).

Accurate species-level identifications underpin many aspects of basic and applied biology; however, identifications can be hampered by a lack of discriminating morphological characters, taxonomic expertise or time. Molecular approaches, such as DNA "barcoding" of the cytochrome c oxidase (COI) gene, are argued to overcome these issues. However, nuclear encoding of mitochondrial genes (numts) and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications. One insect group for which these molecular and morphological problems are significant are the dacine fruit flies (Diptera: Tephritidae: Dacini). We addressed these issues associated with COI barcoding in the dacines by first assessing several "universal" COI primers against public mitochondrial genome and numt sequences for dacine taxa. We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region. Our new primers were tested alongside universal primers on a selection of dacine species, including both fresh preserved and decades-old dry specimens. Additionally, Bactrocera tryoni mitochondrial and nuclear genomes were compared to identify putative numts. Four numt clades were identified, three of which were amplified using existing universal primers. In contrast, our new primers preferentially amplified the "true" mitochondrial COI barcode in all dacine species tested. The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable. Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.

Extensive host-switching of avian feather lice following the Cretaceous-Paleogene mass extinction event.

Nearly all lineages of birds host parasitic feather lice. Based on recent phylogenomic studies, the three major lineages of modern birds diverged from each other before the Cretaceous-Paleogene (K-Pg) mass extinction event. In contrast, studies of the phylogeny of feather lice on birds, indicate that these parasites diversified largely after this event. However, these studies were unable to reconstruct the ancestral avian host lineage for feather lice. Here we use genome sequences of a broad diversity of lice to reconstruct a phylogeny based on 1,075 genes. By comparing this louse evolutionary tree to the avian host tree, we show that feather lice began diversifying on the common ancestor of waterfowl and landfowl, then radiated onto other avian lineages by extensive host-switching. Dating analyses and cophylogenetic comparisons revealed that two of three lineages of birds that diverged before the K-Pg boundary acquired their feather lice after this event via host-switching.

Publisher Correction: Insight into the microbial world of Bemisia tabaci cryptic species complex and its relationships with its host.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Insight into the microbial world of Bemisia tabaci cryptic species complex and its relationships with its host.

The 37 currently recognized Bemisia tabaci cryptic species are economically important species and contain both primary and secondary endosymbionts, but their diversity has never been mapped systematically across the group. To achieve this, PacBio sequencing of full-length bacterial 16S rRNA gene amplicons was carried out on 21 globally collected species in the B. tabaci complex, and two samples from B. afer were used here as outgroups. The microbial diversity was first explored across the major lineages of the whole group and 15 new putative bacterial sequences were observed. Extensive comparison of our results with previous endosymbiont diversity surveys which used PCR or multiplex 454 pyrosequencing platforms showed that the bacterial diversity was underestimated. To validate these new putative bacteria, one of them (Halomonas) was first confirmed to be present in MED B. tabaci using Hiseq2500 and FISH technologies. These results confirmed PacBio is a reliable and informative venue to reveal the bacterial diversity of insects. In addition, many new secondary endosymbiotic strains of Rickettsia and Arsenophonus were found, increasing the known diversity in these groups. For the previously described primary endosymbionts, one Portiera Operational Taxonomic Units (OTU) was shared by all B. tabaci species. The congruence of the B. tabaci-host and Portiera phylogenetic trees provides strong support for the hypothesis that primary endosymbionts co-speciated with their hosts. Likewise, a comparison of bacterial alpha diversities, Principal Coordinate Analysis, indistinct endosymbiotic communities harbored by different species and the co-divergence analyses suggest a lack of association between overall microbial diversity with cryptic species, further indicate that the secondary endosymbiont-mediated speciation is unlikely to have occurred in the B. tabaci species group.

The phylogeny and evolutionary timescale of stoneflies (Insecta: Plecoptera) inferred from mitochondrial genomes.

Phylogenetic analysis based on mitochondrial genomic data from 25 stonefly species recovered a well-supported tree resolving higher-level relationships within Plecoptera (stoneflies). The monophyly of both currently recognized suborders was strongly supported, concordant with previous molecular analyses of Plecoptera. The southern hemisphere suborder Antarctoperlaria formed two clades: Eustheniidae + Diamphipnoidae and Austroperlidae + Gripopterygidae; consistent with relationships proposed based on morphology. The largely northern hemisphere suborder Arctoperlaria also divided into two groups, Euholognatha and Systellognatha, each composed of the five families traditionally assigned to each infraorder (the placement Scopuridae by mt genome data remains untested at this time). Within Euholognatha, strong support for the clade Nemouridae + Notonemouridae confirmed the northern origin of the currently southern hemisphere restricted Notonemouridae. Other family level relationships within the Arctoperlaria differ from those recovered by previous morphology and molecular based analyses. A fossil-calibrated divergence estimation suggests the formation of two suborders dates back to the Jurassic (181 Ma), with subsequent diversification of most stonefly families during the Cretaceous. This result confirms the hypothesis that initial divergence between the suborders was driven by the breakup of the supercontinent Pangaea into Laurasia and Gondwanaland (commencing 200 Ma and complete by 150 Ma).

Phylogenomics and the evolution of hemipteroid insects.

Hemipteroid insects (Paraneoptera), with over 10% of all known insect diversity, are a major component of terrestrial and aquatic ecosystems. Previous phylogenetic analyses have not consistently resolved the relationships among major hemipteroid lineages. We provide maximum likelihood-based phylogenomic analyses of a taxonomically comprehensive dataset comprising sequences of 2,395 single-copy, protein-coding genes for 193 samples of hemipteroid insects and outgroups. These analyses yield a well-supported phylogeny for hemipteroid insects. Monophyly of each of the three hemipteroid orders (Psocodea, Thysanoptera, and Hemiptera) is strongly supported, as are most relationships among suborders and families. Thysanoptera (thrips) is strongly supported as sister to Hemiptera. However, as in a recent large-scale analysis sampling all insect orders, trees from our data matrices support Psocodea (bark lice and parasitic lice) as the sister group to the holometabolous insects (those with complete metamorphosis). In contrast, four-cluster likelihood mapping of these data does not support this result. A molecular dating analysis using 23 fossil calibration points suggests hemipteroid insects began diversifying before the Carboniferous, over 365 million years ago. We also explore implications for understanding the timing of diversification, the evolution of morphological traits, and the evolution of mitochondrial genome organization. These results provide a phylogenetic framework for future studies of the group.

Chromatin immunoprecipitation (ChIP) method for non-model fruit flies (Diptera: Tephritidae) and evidence of histone modifications.

Interactions between DNA and proteins located in the cell nucleus play an important role in controlling physiological processes by specifying, augmenting and regulating context-specific transcription events. Chromatin immunoprecipitation (ChIP) is a widely used methodology to study DNA-protein interactions and has been successfully used in various cell types for over three decades. More recently, by combining ChIP with genomic screening technologies and Next Generation Sequencing (e.g. ChIP-seq), it has become possible to profile DNA-protein interactions (including covalent histone modifications) across entire genomes. However, the applicability of ChIP-chip and ChIP-seq has rarely been extended to non-model species because of a number of technical challenges. Here we report a method that can be used to identify genome wide covalent histone modifications in a group of non-model fruit fly species (Diptera: Tephritidae). The method was developed by testing and refining protocols that have been used in model organisms, including Drosophila melanogaster. We demonstrate that this method is suitable for a group of economically important pest fruit fly species, viz., Bactrocera dorsalis, Ceratitis capitata, Zeugodacus cucurbitae and Bactrocera tryoni. We also report an example ChIP-seq dataset for B. tryoni, providing evidence for histone modifications in the genome of a tephritid fruit fly for the first time. Since tephritids are major agricultural pests globally, this methodology will be a valuable resource to study taxa-specific evolutionary questions and to assist with pest management. It also provides a basis for researchers working with other non-model species to undertake genome wide DNA-protein interaction studies.

Transoceanic Dispersal and Plate Tectonics Shaped Global Cockroach Distributions: Evidence from Mitochondrial Phylogenomics.

Following the acceptance of plate tectonics theory in the latter half of the 20th century, vicariance became the dominant explanation for the distributions of many plant and animal groups. In recent years, however, molecular-clock analyses have challenged a number of well-accepted hypotheses of vicariance. As a widespread group of insects with a fossil record dating back 300 My, cockroaches provide an ideal model for testing hypotheses of vicariance through plate tectonics versus transoceanic dispersal. However, their evolutionary history remains poorly understood, in part due to unresolved relationships among the nine recognized families. Here, we present a phylogenetic estimate of all extant cockroach families, as well as a timescale for their evolution, based on the complete mitochondrial genomes of 119 cockroach species. Divergence dating analyses indicated that the last common ancestor of all extant cockroaches appeared ∼235 Ma, ∼95 My prior to the appearance of fossils that can be assigned to extant families, and before the breakup of Pangaea began. We reconstructed the geographic ranges of ancestral cockroaches and found tentative support for vicariance through plate tectonics within and between several major lineages. We also found evidence of transoceanic dispersal in lineages found across the Australian, Indo-Malayan, African, and Madagascan regions. Our analyses provide evidence that both vicariance and dispersal have played important roles in shaping the distribution and diversity of these insects.

Plant-Mediated Female Transcriptomic Changes Post-Mating in a Tephritid Fruit Fly, Bactrocera tryoni.

Female post-mating behaviors are regulated by complex factors involving males, females, and the environment. In insects, plant secondary compounds that males actively forage for, may indirectly modify female behaviors by altering male behavior and physiology. In the tephritid fruit fly, Bactrocera tryoni, females mated with males previously fed on plant-derived phenylpropanoids (="lures" based on usage in tephritid literature), have longer mating refractoriness, greater fecundity, and reduced longevity than females mated with non-lure fed males. This system thus provides a model for studying transcriptional changes associated with those post-mating behaviors, as the genes regulating the phenotypic changes are likely to be expressed at a greater magnitude than in control females. We performed comparative transcriptome analyses using virgin B. tryoni females, females mated with control males (control-mated), and females mated with lure-fed males (lure-mated). We found 331 differentially expressed genes (DEGs) in control-mated females and 80 additional DEGs in lure-mated females. Although DEGs in control-mated females are mostly immune response genes and chorion proteins, as reported in Drosophila species, DEGs in lure-mated females are titin-like muscle proteins, histones, sperm, and testis expressed proteins which have not been previously reported. While transcripts regulating mating (e.g., lingerer) did not show differential expression in either of the mated female classes, the odorant binding protein Obp56a was down-regulated. The exclusively enriched or suppressed genes in lure-mated females, novel transcripts such as titin and histones, and several taxa-specific transcripts reported here can shed more light on post-mating transcriptional changes, and this can help understand factors possibly regulating female post-mating behaviors.