Absence of the Filarial Endosymbiont Wolbachia in Seal Heartworm (Acanthocheilonema spirocauda) but Evidence of Ancient Lateral Gene Transfer

The symbiotic relationship of Wolbachia spp. was first observed in insects and subsequently in many parasitic filarial nematodes. This bacterium is believed to provide metabolic and developmental assistance to filarial parasitic nematodes, although the exact nature of this relationship remains to be fully elucidated. While Wolbachia is present in most filarial nematodes in the familyOnchocercidae, it is absent in several disparate species such as the human parasite Loa loa. All tested members of the genusAcanthocheilonema, such as Acanthocheilonema viteae, have been shown to lack Wolbachia. Consistent with this, we show thatWolbachia is absent from the seal heartworm (Acanthocheilonema spirocauda), but lateral gene transfer (LGT) of DNA sequences between Wolbachia and A. spirocauda has occurred, indicating a past evolutionary association. Seal heartworm is an important pathogen of phocid seals and understanding its basic biology is essential for conservation of the host. The findings presented here may allow for the development of future treatments or diagnostics for the disease and also aid in clarification of the complicated nematode–Wolbachia relationship

A Novel Quantitative Real-Time PCR Diagnostic Assay for Seal Heartworm (Acanthocheilonema spirocauda) Provides Evidence for Possible Infection in the Grey Seal (Halichoerus grypus)

The distinct evolutionary pressures faced by Pinnipeds have likely resulted in strong coevolutionary ties to their parasites (Leidenberger et al., 2007). This study focuses on the phocid seal filarial heartworm speciesAcanthocheilonema spirocauda. A. spirocauda is known to infect a variety of phocid seals, but does not appear to be restricted to a single host species (Measures et al., 1997; Leidenberger et al., 2007; Lehnert et al., 2015). However, to date, seal heartworm has never been reported in grey seals (Halichoerus grypus) (Measures et al., 1997; Leidenberger et al., 2007; Lehnert et al., 2015). The proposed vector for seal heartworm is Echinophthirius horridus, the seal louse. Seal lice are known to parasitize a wide array of phocid seal species, including the grey seal. With the advent of climate change, disease burden is expected to increase across terrestrial and marine mammals (Harvell et al., 2002). Accordingly, increased prevalence of seal heartworm has recently been reported in harbor seals (Phoca vitulina) (Lehnert et al., 2015). Thus, the need for improved, rapid, and cost-effective diagnostics is urgent. Here we present the first A. spirocauda-specific rapid diagnostic test (a quantitative real- time PCR assay), based on a highly repetitive genomic DNA repeat identified using whole genome sequencing and subsequent bioinformatic analysis. The presence of an insect vector provides the opportunity to develop a multifunctional diagnostic tool that can be used not only to detect the parasite directly from blood or tissue specimens, but also as a molecular xenomonitoring (XM) tool that can be used to assess the epidemiological profile of the parasite by screening the arthropod vector. Using this assay, we provide evidence for the first reported case of seal heartworm in a grey seal

A novel quantitative real-time PCR diagnostic assay for seal heartworm (Acanthocheilonema spirocauda) provides evidence for possible infection in the grey seal (Halichoerus grypus)

The distinct evolutionary pressures faced by Pinnipeds have likely resulted in strong coevolutionary ties to their parasites (Leidenberger et al., 2007). This study focuses on the phocid seal filarial heartworm species Acanthocheilonema spirocauda. A. spirocauda is known to infect a variety of phocid seals, but does not appear to be restricted to a single host species (Measures et al., 1997; Leidenberger et al., 2007; Lehnert et al., 2015). However, to date, seal heartworm has never been reported in grey seals (Halichoerus grypus) (Measures et al., 1997; Leidenberger et al., 2007; Lehnert et al., 2015). The proposed vector for seal heartworm is Echinophthirius horridus, the seal louse. Seal lice are known to parasitize a wide array of phocid seal species, including the grey seal. With the advent of climate change, disease burden is expected to increase across terrestrial and marine mammals (Harvell et al., 2002). Accordingly, increased prevalence of seal heartworm has recently been reported in harbor seals (Phoca vitulina) (Lehnert et al., 2015). Thus, the need for improved, rapid, and cost-effective diagnostics is urgent. Here we present the first A. spirocauda-specific rapid diagnostic test (a quantitative real-time PCR assay), based on a highly repetitive genomic DNA repeat identified using whole genome sequencing and subsequent bioinformatic analysis. The presence of an insect vector provides the opportunity to develop a multifunctional diagnostic tool that can be used not only to detect the parasite directly from blood or tissue specimens, but also as a molecular xenomonitoring (XM) tool that can be used to assess the epidemiological profile of the parasite by screening the arthropod vector. Using this assay, we provide evidence for the first reported case of seal heartworm in a grey seal. Keywords: Harbor seal, Parasites, Seal heartworm, Acanthocheilonema spirocauda, Real-time PCR, Molecular diagnostics, Xenomonitoring, Genomic repeat

A Novel Quantitative Real-Time PCR Diagnostic Assay for Fecal and Nasal Swab Detection of an Otariid Lungworm, \u3ci\u3eParafilaroides decorus\u3c/i\u3e

Parafilaroides decorus, also known as sea lion lungworm, is a metastrongyloid nematode that infects otariid hosts, such as the charismatic California sea lion, Zalophus californianus. P. decorus causes bronchointerstitial pneumonia, respiratory distress, reduced ability to swim, dive and hunt and as a result, increased mortality particularly in young animals. Respiratory disease is a leading cause of stranding and admission to rehabilitation centers on the Pacific coast. Low-coverage genomic sequencing of four P. decorus individuals analyzed through Galaxy\u27s RepeatExplorer identified a novel repeat DNA family we employed to design a sensitive quantitative PCR (qPCR) assay for diagnosing infections from fecal or sputum samples. The assay detects as little as 10 fg of P. decorus DNA and a linear regression model developed using a standard curve can be used to estimate the concentration of P. decorus DNA in a sample, ± 0.015 ng. This knowledge can be leveraged to estimate the level of parasite burden, which can be used to design improved treatments for animals in rehabilitation. Improved treatment of infections will aid in more animals being successfully released back into the wild

Differential expression and Gene Ontology enrichment analysis (GOEA).

(A) Total number of inferred replication cycle differentially expressed genes (DEGs) in each species independent of other species. (B) Stack bar plot showing proportion of DEGs in each phase in all species. (C) Bar plot showing the total number of conserved DEGs of inferred replication cycle phases (rows) across all species. (D) Total number of DEGs of the indicated phase unique to the indicated species (colors). The most significant Gene Ontology (GO) term (Benjamini 2 and adjusted p-value S3 and S4 Tables. Data and code for generating the figure are available at https://github.com/umbibio/scBabesiaAtlases.</p

Quantification of alignment of gene curves between synchronized bulk and single-cell sequencing.

(A) Dynamic time warping alignment of a sample gene: high alignments (left), low alignments (right). Gray dashed lines indicate the matched time index (warping). (B) Top left: Correlation between calculated peak times in S/M/C phases, excluding the boundary. Top right: Percent alignment distance categorized into high (distance upper 80th percentile), and mid. Curves with no peak contain a higher percentage of poor alignment. Bottom: Distribution of dynamic time warping (dtw) alignment distance between single-cell and bulk sequencing. Shaded areas show boundaries of alignment categories. NA, not available. Data and code for generating the figure are available at https://github.com/umbibio/scBabesiaAtlases.</p

Projection of single-cell RNA sequencing data onto UMAP coordinates and the first 3 PC coordinates.

B. divergens in human and bovine host cells cluster together. PCA, principal component; UMAP, Uniform Manifold Approximation and Projection. Data and code for generating the figure are available at https://github.com/umbibio/scBabesiaAtlases.</p

Differential expression.

Tabs 1–5 contain the list of differentially expressed genes (DEGs) of the inferred cell cycle phases (FC > 2, adjusted p-value B. divergens and species-independent DEGs in the human versus bovine comparison. (XLSX)</p

Additional figures.

S1 Text also contains extended analysis on species-specific markers and the human versus bovine marker analysis. (PDF)</p

Co-expression network.

(A) Figure showing the number of overlapping genes in the indicated contrast. (B) The expression curves of the 10 conserved hub genes at the intersection grouped into 2 clusters with similar expression profile. (C) The inferred interactome of 10 conserved hub genes in the B. divergens (human) co-expression network. NA, not available. Data and code for generating the figure are available at https://github.com/umbibio/scBabesiaAtlases.</p