Developmental and comparative immunology single-cell transcriptome analysis of the B-cell repertoire reveals the usage of immunoglobulins in the gray short-tailed opossum (Monodelphis domestica).

B-cells are key to humoral immunity, are found in multiple lymphoid organs, and have the unique ability to mediate the production of antigen-specific antibodies in the presence of pathogens. The marsupial immunoglobulin (Ig) heavy (H) chain locus encodes four constant region isotypes, IgA, IgG, IgM and IgE, but no IgD, and there are two light (L) chain isotypes, lambda (Igλ) and kappa (Igκ). To gain an understanding of the marsupial humoral immune system, B-cell transcriptomes generated by single-cell RNA sequencing from gray short-tailed opossum (Monodelphis domestica) splenocytes, and peripheral blood mononuclear cells were analysed. The cells used were from a single unimmunized animal and the majority of B-cells were transcribing IgM heavy chains. The ratio of Ig light chain use was roughly 2:1, Igλ:Igκ in this individual. This was not predicted due to Igκ being the more complex of the two L chain loci. The variable (V) gene segment pairs used in individual B-cells confirm greater diversity provided by the L chain V. This study is the first to report on using single cell analysis to investigate Ig repertoires in a marsupial and confirms a number of prior hypothesis, as well as revealing some surprises.

The environmental and ecological determinants of elevated Ross River Virus exposure in koalas residing in urban coastal landscapes.

Koala populations in many areas of Australia have declined sharply in response to habitat loss, disease and the effects of climate change. Koalas may face further morbidity from endemic mosquito-borne viruses, but the impact of such viruses is currently unknown. Few seroprevalence studies in the wild exist and little is known of the determinants of exposure. Here, we exploited a large, spatially and temporally explicit koala survey to define the intensity of Ross River Virus (RRV) exposure in koalas residing in urban coastal environments in southeast Queensland, Australia. We demonstrate that RRV exposure in koalas is much higher (> 80%) than reported in other sero-surveys and that exposure is uniform across the urban coastal landscape. Uniformity in exposure is related to the presence of the major RRV mosquito vector, Culex annulirostris, and similarities in animal movement, tree use, and age-dependent increases in exposure risk. Elevated exposure ultimately appears to result from the confinement of remaining coastal koala habitat to the edges of permanent wetlands unsuitable for urban development and which produce large numbers of competent mosquito vectors. The results further illustrate that koalas and other RRV-susceptible vertebrates may serve as useful sentinels of human urban exposure in endemic areas.

Mosquito-Borne Viruses and Non-Human Vertebrates in Australia: A Review.

Mosquito-borne viruses are well recognized as a global public health burden amongst humans, but the effects on non-human vertebrates is rarely reported. Australia, houses a number of endemic mosquito-borne viruses, such as Ross River virus, Barmah Forest virus, and Murray Valley encephalitis virus. In this review, we synthesize the current state of mosquito-borne viruses impacting non-human vertebrates in Australia, including diseases that could be introduced due to local mosquito distribution. Given the unique island biogeography of Australia and the endemism of vertebrate species (including macropods and monotremes), Australia is highly susceptible to foreign mosquito species becoming established, and mosquito-borne viruses becoming endemic alongside novel reservoirs. For each virus, we summarize the known geographic distribution, mosquito vectors, vertebrate hosts, clinical signs and treatments, and highlight the importance of including non-human vertebrates in the assessment of future disease outbreaks. The mosquito-borne viruses discussed can impact wildlife, livestock, and companion animals, causing significant changes to Australian ecology and economy. The complex nature of mosquito-borne disease, and challenges in assessing the impacts to non-human vertebrate species, makes this an important topic to periodically review.

The entomological impact of passive metofluthrin emanators against indoor Aedes aegypti: A randomized field trial.

BACKGROUND: In the absence of vaccines or drugs, insecticides are the mainstay of Aedes-borne disease control. Their utility is challenged by the slow deployment of resources, poor community compliance and inadequate household coverage. Novel application methods are required. METHODOLOGY AND PRINCIPAL FINDINGS: A 10% w/w metofluthrin "emanator" that passively disseminates insecticide from an impregnated net was evaluated in a randomized trial of 200 houses in Mexico. The devices were introduced at a rate of 1 per room and replaced at 3-week intervals. During each of 7 consecutive deployment cycles, indoor resting mosquitoes were sampled using aspirator collections. Assessments of mosquito landing behaviours were made in a subset of houses. Pre-treatment, there were no differences in Aedes aegypti indices between houses recruited to the control and treatment arms. Immediately after metofluthrin deployment, the entomological indices between the trial arms diverged. Averaged across the trial, there were significant reductions in Abundance Rate Ratios for total Ae. aegypti, female abundance and females that contained blood meals (2.5, 2.4 and 2.3-times fewer mosquitoes respectively; P<0.001). Average efficacy was 60.2% for total adults, 58.3% for females, and 57.2% for blood-fed females. The emanators also reduced mosquito landings by 90% from 12.5 to 1.2 per 10-minute sampling period (P<0.05). Homozygous forms of the pyrethroid resistant kdr alleles V410L, V1016L and F1534C were common in the target mosquito population; found in 39%, 24% and 95% of mosquitoes collected during the trial. CONCLUSIONS/SIGNIFICANCE: This is the first randomized control trial to evaluate the entomological impact of any volatile pyrethroid on urban Ae. aegypti. It demonstrates that volatile pyrethroids can have a sustained impact on Ae. aegypti population densities and human-vector contact indoors. These effects occur despite the presence of pyrethroid-resistant alleles in the target population. Formulations like these may have considerable utility for public health vector control responses.

Red-tailed phascogales: A review of their biology and importance as model marsupial species.

There are many limitations when using traditional laboratory species. Limits on variation, may result in limited outcomes, at both the species and individual level, due to different individuals/species having diverse physiological processes, or differing molecular and genetic mechanisms. By using a variety of model species, we will be able to develop creative solutions to biological problems and identify differences of which we were not previously aware. The laboratory mouse has been a suitable model species for various mammalian studies, however most are bred specifically for laboratory research with limited variability due to selective breeding. Marsupial models offer unique research opportunities compared to eutherian models. We believe that there should be an expansion in marsupial model species, and the introduction of the red-tailed phascogale (Phascogale calura), a dasyurid marsupial, should be one of them. Phascogales are easily managed in captivity, and there are now multiple studies involving their development, reproduction, nutrition, behavior and immune system, which can serve as a baseline for future studies. The addition of the phascogale as a model species will improve future mammalian studies by introducing variability and offer alternate solutions to biological problems, particularly in the areas of genetics, nutrition, immunology, the neuro-endocrine system, and ageing, due to their semelparous reproductive strategy and hence, subsequent predictive physiology. In this review, we provide information based on existing research on red-tailed phascogales to support their inclusion as a model species.

Ability of near-infrared spectroscopy and chemometrics to predict the age of mosquitoes reared under different conditions.

BACKGROUND: Practical, field-ready age-grading tools for mosquito vectors of disease are urgently needed because of the impact that daily survival has on vectorial capacity. Previous studies have shown that near-infrared spectroscopy (NIRS), in combination with chemometrics and predictive modeling, can forecast the age of laboratory-reared mosquitoes with moderate to high accuracy. It remains unclear whether the technique has utility for identifying shifts in the age structure of wild-caught mosquitoes. Here we investigate whether models derived from the laboratory strain of mosquitoes can be used to predict the age of mosquitoes grown from pupae collected in the field. METHODS: NIRS data from adult female Aedes albopictus mosquitoes reared in the laboratory (2, 5, 8, 12 and 15 days-old) were analysed against spectra from mosquitoes emerging from wild-caught pupae (1, 7 and 14 days-old). Different partial least squares (PLS) regression methods trained on spectra from laboratory mosquitoes were evaluated on their ability to predict the age of mosquitoes from more natural environments. RESULTS: Models trained on spectra from laboratory-reared material were able to predict the age of other laboratory-reared mosquitoes with moderate accuracy and successfully differentiated all day 2 and 15 mosquitoes. Models derived with laboratory mosquitoes could not differentiate between field-derived age groups, with age predictions relatively indistinguishable for day 1-14. Pre-processing of spectral data and improving the PLS regression framework to avoid overfitting can increase accuracy, but predictions of mosquitoes reared in different environments remained poor. Principal components analysis confirms substantial spectral variations between laboratory and field-derived mosquitoes despite both originating from the same island population. CONCLUSIONS: Models trained on laboratory mosquitoes were able to predict ages of laboratory mosquitoes with good sensitivity and specificity though they were unable to predict age of field-derived mosquitoes. This study suggests that laboratory-reared mosquitoes do not capture enough environmental variation to accurately predict the age of the same species reared under different conditions. Further research is needed to explore alternative pre-processing methods and machine learning techniques, and to understand factors that affect absorbance in mosquitoes before field application using NIRS.

First evidence of concurrent enzootic and endemic transmission of Ross River virus in the absence of marsupial reservoirs in Fiji.

BACKGROUND: Ross River virus (RRV) is a zoonotic alphavirus transmitted by several mosquito species. Until recently, endemic transmission was only considered possible in the presence of marsupial reservoirs. METHODS: RRV seroprevalence was investigated in placental mammals (including horses, cows, goats, pigs, dogs, rats, and mice) in Fiji, where there are no marsupials. A total of 302 vertebrate serum samples were collected from 86 households from 10 communities in Western Fiji. RESULTS: Neutralizing antibodies against RRV were detected in 28% to 100% of sera depending on the species, and neutralization was strong even at high dilutions. CONCLUSIONS: These results are unlikely to be due to cross-reactions. Chikungunya is the only other alphavirus known to be present in the Pacific Islands, but it rarely spills over into non-humans, even during epidemics. The study findings, together with a recent report of high RRV seroprevalence in humans, strongly suggest that RRV is circulating in Fiji in the absence of marsupial reservoirs. Considering that all non-human vertebrates present in Fiji are pan-global in distribution, RRV has the potential to further expand its geographic range. Further surveillance of RRV and access to RRV diagnostics will be critical for the early detection of emergence and outbreaks.

Mosquito Age Grading and Vector-Control Programmes.

An ability to characterize the age of mosquito populations could provide cost-effective and compelling entomological evidence for the potential epidemiological impacts of vector control. The average age of a mosquito population is the most important determinant of vectorial capacity and the likelihood of disease transmission. Yet, despite decades of research, defining the age of a wild-caught mosquito remains a challenging, impractical, and unreliable process. Emerging chemometric and existing transcriptional approaches may overcome many of the limitations of current morphological techniques, but their utility in terms of field-based monitoring programmes remains largely untested. Herein, we review the potential advantages and disadvantages of new and existing age-grading tools in an operational context.

Immunogenetics of marsupial B-cells.

Marsupials and eutherians are mammals that differ in their physiological traits, predominately their reproductive and developmental strategies; eutherians give birth to well-developed young, while marsupials are born highly altricial after a much shorter gestation. These developmental traits also result in differences in the development of the immune system of eutherian and marsupial species. In eutherians, B-cells are the key to humoral immunity as they are found in multiple lymphoid organs and have the unique ability to mediate the production of antigen-specific antibodies in the presence of extracellular pathogens. The development of B-cells in marsupials has been reported and hypothesised to be similar to that of eutherians, except that haematopoiesis occurs in the liver, postpartum, until the bone marrow fully matures. In eutherians, specific genes are linked to specific stages in B-cell development, maturation, and differentiation processes, and have been identified including immunoglobulins (heavy and light chains), cluster of differentiation markers (CD10, 19, 34 and CD79α/ß), signal transduction molecules (BTK, Lyn and Syk) and transcriptional regulators (EBF1, E2A, and Pax5). This review aims to discuss the known similarities and differences between marsupial and eutherian B-cells, in regards to their genetic presence, homology, and developmental stages, as well as to highlight the areas requiring further investigation. By enhancing our understanding of the genes that are involved with B-cells in the marsupial lineage, it will, in turn, aid our understanding of the marsupial immune system and support the development of specific immunological reagents for research and wildlife conservation purposes.

Treatment of pigs with endectocides as a complementary tool for combating malaria transmission by Anopheles farauti (s.s.) in Papua New Guinea.

BACKGROUND: Outdoor, early-biting, zoophagic behaviours by Anopheles farauti (s.s.) can compromise the effectiveness of bed nets for malaria control. In the Western Pacific region, pigs and dogs represent significant alternative blood sources for mosquitoes. Treating these animals with endectocides may impact mosquito survival and complement control measures. This hypothesis was explored using membrane feeding assays (MFAs), direct feeds on treated pigs, pharmacokinetic analyses and a transmission model. RESULTS: Ivermectin was 375-fold more mosquitocidal than moxidectin (24 h LC50 = 17.8 ng/ml vs 6.7 µg/ml) in MFAs, and reduced mosquito fecundity by > 50% at ≥ 5 ng/ml. Treatment of pigs with subcutaneous doses of 0.6 mg/kg ivermectin caused 100% mosquito mortality 8 days after administration. Lethal effects persisted for up to 15 days after administration (75% death within 10 days). CONCLUSION: The application of these empirical data to a unique malaria transmission model that used a three-host system (humans, pigs and dogs) predicts that the application of ivermectin will cause a significant reduction in the entomological inoculation rate (EIR = 100 to 0.35). However, this is contingent on local malaria vectors sourcing a significant proportion of their blood meals from pigs. This provides significant insights on the benefits of deploying endectocides alongside long-lasting insecticide-treated nets (LLINs) to address residual malaria transmission.

Antimicrobial activity of red-tailed phascogale (Phascogale calura) serum.

Antimicrobial substances in serum include circulating complement proteins and acute phase proteins (APPs). We identified gene sequences for APPs, haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA) in marsupial genomes. Hp and SAA levels were measured in red-tailed phascogale (Phascogale calura) sera using commercially available assays. Hp levels were higher in males than females, while SAA levels suggest the phascogales used in this study were healthy. Serum was co-cultured with four bacterial species. Bacterial growth was inhibited after incubation at 37°C, however effectiveness differed with bacteria and incubation time. The least amount of bacterial growth was noticed after introduction to K. pneumoniae, and most when introduced to P. aeruginosa. Despite marsupials not having mature immune tissues at birth, and unable to mount specific immune responses, this study suggests other immune strategies, such as APPs in serum likely aid marsupials in their defence against pathogens.

Evaluation of reference genes for gene expression in red-tailed phascogale (Phascogale calura) liver, lung, small intestine and spleen.

BACKGROUND: Reference genes serve an important role as an endogenous control/standard for data normalisation in gene expression studies. Although reference genes have recently been suggested for marsupials, independent analysis of reference genes on different immune tissues is yet to be tested. Therefore, an assessment of reference genes is needed for the selection of stable, expressed genes across different marsupial tissues. METHODS: The study was conducted on red-tailed phascogales (Phascogale calura) using five juvenile and five adult males. The stability of five reference genes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH; ß-actin, ACTB; 18S rRNA, 18S; 28S rRNA, 28S; and ribosomal protein L13A, RPL13A) was investigated using SYBR Green and analysed with the geNorm application available in qBasePLUS software. RESULTS: Gene stability for juvenile and adult tissue samples combined show that GAPDH was most stable in liver and lung tissue, and 18S in small intestine and spleen. While all reference genes were suitable for small intestine and spleen tissues, all reference genes except 28S were stable for lung and only 18S and 28S were stable for liver tissue. Separating the two age groups, we found that two different reference genes were considered stable in juveniles (ACTB and GAPDH) and adults (18S and 28S), and RPL13A was not stable for juvenile small intestine tissue. Except for 28S, all reference genes were stable in juvenile and adult lungs, and all five reference genes were stable in spleen tissue. DISCUSSION: Based on expression stability, ACTB and GAPDH are suitable for all tissues when studying the expression of marsupials in two age groups, except for adult liver tissues. The expression stability between juvenile and adult liver tissue was most unstable, as the stable reference genes for juveniles and adults were different. Juvenile and adult lung, small intestine and spleen share similar stable reference genes, except for small intestine tissues where all reference genes were stable in adults but RPL13A was not suitable in juveniles.

Sequences and expression of pathway-specific complement components in developing red-tailed phascogale (Phascogale calura).

Marsupials are born immunologically premature, relying on cells and molecules in maternal milk for immune protection. Both immunoglobulin and complement proteins have been identified in marsupial milk, but the expression of specific complement proteins remains largely unexplored. We report partial cDNA sequences for two complement-activating proteins, C3, C1r, CFP and MASP2, in liver tissues from red-tailed phascogale (Phascogale calura). Conservation of functionally relevant motifs were identified in the translated cDNA sequences from phascogale C3, CFP and MASP2 and their eutherian homologues. Gene expression of representative molecules from each of the major complement pathways was also investigated in whole body tissues from 1 to 18 day old animals and liver tissues from 31-day to 14-month old animals. Average complement expression in whole bodies and liver tissues of C1r, CFP, MASP2 and C3 increased significantly in juveniles compared to pouch young, presumably due to the maturation of the young's own complement system. Comparing expression in liver tissues only, we found that the average CFP expression were higher in pouch young compared to juveniles, while results were still statistically similar to the average expression of all tissues for C1r, MASP2 and C3. The average complement expression then significantly decreased as the animals aged into adulthood.