Benchmarking assembly free nanopore read mappers to classify complex millipede gut microbiota via Oxford Nanopore Sequencing Technology.

Millipedes are key players in recycling leaf litter into soil in tropical ecosystems. To elucidate their gut microbiota, we collected millipedes from different municipalities of Puerto Rico. Here we aim to benchmark which method is best for metagenomic skimming of this highly complex millipede microbiome. We sequenced the gut DNA with Oxford Nanopore Technologies' (ONT) MinION sequencer, then analyzed the data using MEGAN-LR, Kraken2 protein mode, Kraken2 nucleotide mode, GraphMap, and Minimap2 to classify these long ONT reads. From our two samples, we obtained a total of 87,110 and 99,749 ONT reads, respectively. Kraken2 nucleotide mode classified the most reads compared to all other methods at the phylum and class taxonomic level, classifying 75% of the reads in the two samples, the other methods failed to assign enough reads to either phylum or class to yield asymptotes in the taxa rarefaction curves indicating that they required more sequencing depth to fully classify this community. The community is hyper diverse with all methods classifying 20-50 phyla in the two samples. There was significant overlap in the reads used and phyla classified between the five methods benchmarked. Our results suggest that Kraken2 nucleotide mode is the most appropriate tool for the application of metagenomic skimming of this highly complex community.

Metagenomic binning of PacBio HiFi data prior to assembly reveals a complete genome of Cosmopolites sordidus (Germar) (Coleopterea: Curculionidae, Dryophthorinae) the most damaging arthropod pest of bananas and plantains.

PacBio HiFi sequencing was employed in combination with metagenomic binning to produce a high-quality reference genome of Cosmopolites sordidus. We compared k-mer and alignment reference based pre-binning and post-binning approaches to remove contamination. We were also interested to know if the post-binning approach had interspersed bacterial contamination within intragenic regions of Arthropoda binned contigs. Our analyses identified 3,433 genes that were composed with reads identified as of putative bacterial origins. The pre-binning approach yielded a C. sordidus genome of 1.07 Gb genome composed of 3,089 contigs with 98.6% and 97.1% complete and single copy genome and protein BUSCO scores respectively. In this article we demonstrate that in this case the pre-binning approach does not sacrifice assembly quality for more stringent metagenomic filtering. We also determine post-binning allows for increased intragenic contamination increased with increasing coverage, but the frequency of gene contamination increased with lower coverage. Future work should focus on developing reference free pre-binning approaches for HiFi reads produced from eukaryotic based metagenomic samples.

Metagenomic clustering reveals microbial contamination as an essential consideration in ultraconserved element design for phylogenomics with insect museum specimens.

Phylogenomics via ultraconserved elements (UCEs) has led to improved phylogenetic reconstructions across the tree of life. However, inadvertently incorporating non-targeted DNA into the UCE marker design will lead to misinformation being incorporated into subsequent analyses. To date, the effectiveness of basic metagenomic filtering strategies has not been assessed in arthropods. Designing markers from museum specimens requires careful consideration of methods due to the high levels of microbial contamination typically found in such specimens. We investigate if contaminant sequences are carried forward into a UCE marker set we developed from insect museum specimens using a standard bioinformatics pipeline. We find that the methods currently employed by most researchers do not exclude contamination from the final set of targets. Lastly, we highlight several paths forward for reducing contamination in UCE marker design.

Intercontinental distributions, phylogenetic position and life cycles of species of Apharyngostrigea (Digenea, Diplostomoidea) illuminated with morphological, experimental, molecular and genomic data.

When subjected to molecular study, species of digeneans believed to be cosmopolitan are usually found to consist of complexes of species with narrower distributions. We present molecular and morphological evidence of transcontinental distributions in two species of Apharyngostrigea Ciurea, 1924, based on samples from Africa and the Americas. Sequences of cytochrome c oxidase I and, in some samples, internal transcribed spacer, revealed Apharyngostrigea pipientis (Faust, 1918) in Tanzania (first known African record), Argentina, Brazil, USA and Canada. Sequences from A. pipientis also match previously published sequences identified as Apharyngostrigea cornu (Zeder, 1800) originating in Mexico. Hosts of A. pipientis surveyed include definitive hosts from the Afrotropic, Neotropic and Nearctic, as well as first and second intermediate hosts from the Americas, including the type host and type region. In addition, metacercariae of A. pipientis were obtained from experimentally infected Poecilia reticulata, the first known record of this parasite in a non-amphibian second intermediate host. Variation in cytochrome c oxidase I haplotypes in A. pipientis is consistent with a long established, wide-ranging species with moderate genetic structure among Nearctic, Neotropic and Afrotropic regions. We attribute this to natural dispersal by birds and find no evidence of anthropogenic introductions of exotic host species. Sequences of CO1 and ITS from adult Apharyngostrigea simplex (Johnston, 1904) from Egretta thula in Argentina matched published data from cercariae from Biomphalaria straminea from Brazil and metacercariae from Cnesterodon decemmaculatus in Argentina, consistent with previous morphological and life-cycle studies reporting this parasite-originally described in Australia-in South America. Analyses of the mitochondrial genome and rDNA operon from A. pipientis support prior phylogenies based on shorter markers showing the Strigeidae Railliet, 1919 to be polyphyletic.

Validity of the Diplostomoidea and Diplostomida (Digenea, Platyhelminthes) upheld in phylogenomic analysis.

Higher systematics within the Digenea, Carus 1863 have been relatively stable since a phylogenetic analysis of partial nuclear ribosomal markers (rDNA) led to the erection of the Diplostomida Olson, Cribb, Tkach, Bray, and Littlewood, 2003. However, recent mitochondrial (mt) genome phylogenies suggest this order might be paraphyletic. These analyses show members of two diplostomidan superfamilies are more closely related to the Plagiorchiida La Rue, 1957 than to other members of the Diplostomida. A recent phylogeny based on partial cytochrome c oxidase I also indicates one of the groups implicated, the Diplostomoidea Poirier, 1886, is non-monophyletic. To determine if these results were robust to additional taxon sampling, we analyzed mt genomes from seven diplostomoids in three families. To choose between phylogenetic alternatives based on mt genomes and the prior rDNA-based topology, we analyzed hundreds of ultra-conserved genomic elements assembled from shotgun sequencing. The Diplostomida was paraphyletic in the mt genome phylogeny but supported in the ultra-conserved genomic element phylogeny. We speculate this mitonuclear discordance is related to ancient, rapid radiation in the Digenea. Both ultra-conserved genomic elements and mt genomes support the monophyly of the Diplostomoidea and show congruent relationships within it. The Cyathocotylidae Mühling, 1898 are early diverging descendants of a paraphyletic clade of Diplostomidae Poirier, 1886, in which are nested members of the Strigeidae Railliet, 1919; the results support prior suggestions that the Crassiphialinae Sudarikov, 1960 will rise to the family level. Morphological traits of diplostomoid metacercariae appear to be more useful for differentiating clades than those of adults. We describe a new species of Cotylurus Szidat, 1928, resurrect a species of Hysteromorpha Lutz, 1931, and find support for a species of Alaria Schrank, 1788 of contested validity. Complete rDNA operons from seven diplostomoid species are provided as a resource for future studies.

On the biosynthetic origin of carminic acid.

The chemical composition of the scale insect Dactylopius coccus was analyzed with the aim to discover new possible intermediates in the biosynthesis of carminic acid. UPLC-DAD/HRMS analyses of fresh and dried insects resulted in the identification of three novel carminic acid analogues and the verification of several previously described intermediates. Structural elucidation revealed that the three novel compounds were desoxyerythrolaccin-O-glucosyl (DE-O-Glcp), 5,6-didehydroxyerythrolaccin 3-O-ß-D-glucopyranoside (DDE-3-O-Glcp), and flavokermesic acid anthrone (FKA). The finding of FKA in D. coccus provides solid evidence of a polyketide, rather than a shikimate, origin of coccid pigments. Based on the newly identified compounds, we present a detailed biosynthetic scheme that accounts for the formation of carminic acid (CA) in D. coccus and all described coccid pigments which share a flavokermesic acid (FK) core. Detection of coccid pigment intermediates in members of the Planococcus (mealybugs) and Pseudaulacaspis genera shows that the ability to form these pigments is taxonomically more widely spread than previously documented. The shared core-FK-biosynthetic pathway and wider taxonomic distribution suggests a common evolutionary origin for the trait in all coccid dye producing insect species.

Gene expression plasticity across hosts of an invasive scale insect species.

For plant-eating insects, we still have only a nascent understanding of the genetic basis of host-use promiscuity. Here, to improve that situation, we investigated host-induced gene expression plasticity in the invasive lobate lac scale insect, Paratachardina pseudolobata (Hemiptera: Keriidae). We were particularly interested in the differential expression of detoxification and effector genes, which are thought to be critical for overcoming a plant's chemical defenses. We collected RNA samples from P. pseudolobata on three different host plant species, assembled transcriptomes de novo, and identified transcripts with significant host-induced gene expression changes. Gene expression plasticity was pervasive, but the expression of most detoxification and effector genes was insensitive to the host environment. Nevertheless, some types of detoxification genes were more differentially expressed than expected by chance. Moreover, we found evidence of a trade-off between expression of genes involved in primary and secondary metabolism; hosts that induced lower expression of genes for detoxification induced higher expression of genes for growth. Our findings are largely consonant with those of several recently published studies of other plant-eating insect species. Thus, across plant-eating insect species, there may be a common set of gene expression changes that enable host-use promiscuity.

Early divergent strains of Yersinia pestis in Eurasia 5,000 years ago.

The bacteria Yersinia pestis is the etiological agent of plague and has caused human pandemics with millions of deaths in historic times. How and when it originated remains contentious. Here, we report the oldest direct evidence of Yersinia pestis identified by ancient DNA in human teeth from Asia and Europe dating from 2,800 to 5,000 years ago. By sequencing the genomes, we find that these ancient plague strains are basal to all known Yersinia pestis. We find the origins of the Yersinia pestis lineage to be at least two times older than previous estimates. We also identify a temporal sequence of genetic changes that lead to increased virulence and the emergence of the bubonic plague. Our results show that plague infection was endemic in the human populations of Eurasia at least 3,000 years before any historical recordings of pandemics.

Distribution of Culex species in vegetation bands of a constructed wetland undergoing integrated mosquito management.

The distribution and abundance of emerging Culex spp. were assessed within narrow (width: 3 m) and wide (width: 20 m) bands of California bulrush (Schoenoplectus californicus) and in the open water adjacent to emergent vegetation in 2 marshes of an ammonia-dominated wastewater treatment wetland in southern California. Emerging mosquitoes were collected along transects perpendicular to the path of water flow at 3 distances (1.5, 5, and 10 m) from the vegetation-open water interface in the wide bands of emergent vegetation, at the center of narrow bands of emergent vegetation, and at 1.5 m from the edge of emergent vegetation in the open water. The width of vegetation bands (3 vs. 20 m) influenced the effectiveness of integrated mosquito management practices, especially the application of mosquito control agents. Mosquito production from the 2 marshes also differed up to 14-fold, suggesting that the distance between the shorelines (62 vs. 74 m) of each marsh also influenced the efficacy of mosquito control agents applied from the shore and boats. Hot spots of mosquito production (75424 female Culex/m2/day) were found within the wide bands of bulrush. During summer, the relative abundance of Culex stigmatosoma among emerging mosquitoes increased from the periphery to the center of wide bands of emergent vegetation. Culex erythrothorax emergence rates were comparatively similar among the transects in the wide bands of emergent vegetation. Culex tarsalis adults increased in number from the periphery to the center of wide bands of bulrush and, in May, were > 95% of emerged mosquitoes.

Ovipositional responses of two Culex (Diptera: Culicidae) species to larvivorous fish.

Species-specific differences in the responses of egg-laying mosquitoes (Diptera: Culicidae) to the presence of fish exudates in oviposition sites in laboratory bioassays have been related to the likelihood of encountering mosquito-eating fish in natural oviposition sites. We examined the responses of egg-laying mosquitoes to the presence of larvivorous fish in oviposition sites to test this hypothesis in the field. The number of Culex tarsalis Coquillett egg rafts laid on mesocosms (15.5 m2; 8 m3) containing caged mosquitofish (0.066 Gambusia affinis per liter) was reduced by 84% relative to mesocosms lacking fish. Egg-laying Culex quinquefasciatus Say did not differentiate significantly between comparatively small (0.11 m2) oviposition sites containing water conditioned with mosquitofish (0.3 G. affinis per liter) versus aged reservoir water. Cx. quinquefasciatus egg rafts were not collected from the larger mesocosms, and Cx. tarsalis egg rafts were rarely collected from the smaller oviposition sites. Oviposition preferences for characteristics of aquatic habitats that lack fish (e.g., small size, semipermanence, and high levels of organic enrichment in which hypoxic conditions are prevalent) may limit the coexistence of immature stages of some mosquito species such as Cx. quinquefasciatus and insectivorous fish. Mosquito species such as Cx. tarsalis that also use comparatively large water bodies as developmental sites may have evolved the ability to detect the presence of predatory fish.

Comparison of mosquito control provided by the arroyo chub (Gila orcutti) and the mosquitofish (Gambusia affinis).

Two 6-wk trials were conducted in 28-m2 earthen ponds to compare the efficacy of the arroyo chub, Gila orcutti, to the mosquitofish, Gambusia affinis, as a biological control agent for mosquitoes and a possible replacement for the mosquitofish in sensitive watersheds of southern California. The mosquitofish population growth rate was 1.73 times greater than the arroyo chub population growth rate; however, greater reproduction by the mosquitofish did not result in significantly better reduction of mosquitoes than was provided by the comparatively small populations of arroyo chub. On average across a 6-wk study in the spring, both larvivorous fishes reduced the abundance of 3rd and 4th instars by 4- to 5-fold compared to that observed in the control ponds that lacked fish but contained few invertebrate predators. The abundance of nontarget microinvertebrates in ponds containing the mosquitofish was only 7% of that in ponds containing the arroyo chub during the summer, but did not differ significantly between the fish species treatments when zooplankton was comparatively more abundant during the spring. Even though the number of individuals produced by each fish species during 6 wk in the spring was greater than for fish stocked in the summer, species-specific population growth rates in the spring study (individuals/individual/d; mosquitofish, 0.077; arroyo chub, 0.044) were only slightly higher than in the summer (individuals/individual/d; mosquitofish, 0.068; arroyo chub, 0.039) indicating that differences in the number of fish stocked contributed primarily to the differences in final population size between spring and summer studies. The arroyo chub is native to the South Coastal drainages in California and should be considered as a viable alternative to the mosquitofish for integrated mosquito management programs in riverine wetlands and sensitive watersheds of southern California.