Wolbachia are maternally inherited bacterial endosymbionts that naturally infect a diverse array of arthropods. They are primarily known for their manipulation of host reproductive biology, and recently, infections with Wolbachia have been proposed as a new strategy for controlling insect vectors and subsequent human-transmissible diseases. Yet, Wolbachia abundance has been shown to vary greatly between individuals and the magnitude of the effects of infection on host life-history traits and protection against infection is correlated to within-host Wolbachia abundance. It is therefore essential to better understand the factors that modulate Wolbachia abundance and effects on host fitness. Nutrition is known to be one of the most important mediators of host-symbiont interactions. Here, we used nutritional geometry to quantify the role of macronutrients on insect-Wolbachia relationships in Drosophila melanogaster. Our results show fundamental interactions between diet composition, host diet selection, Wolbachia abundance and effects on host lifespan and fecundity. The results and methods described here open a new avenue in the study of insect-Wolbachia relationships and are of general interest to numerous research disciplines, ranging from nutrition and life-history theory to public health.

Aedes mosquitoes harboring intracellular Wolbachia bacteria are being released in arbovirus and mosquito control programs. With releases taking place around the world, understanding the contribution of host variation to Wolbachia phenotype is crucial. We generated a Wolbachia transinfection ( w AlbB Q ) in Aedes aegypti and performed backcrossing to introduce the infection into Australian or Malaysian nuclear backgrounds. Whole Wolbachia genome sequencing shows that the w AlbB Q transinfection is near-identical to the reference w AlbB genome, suggesting few changes since the infection was first introduced to Ae. aegypti over 15 years ago. However, these sequences were distinct from other available w AlbB genome sequences, highlighting the potential diversity of w AlbB in natural Ae. albopictus populations. Phenotypic comparisons demonstrate effects of w AlbB infection on egg hatch and nuclear background on fecundity and body size, but no interactions between w AlbB infection and nuclear background for any trait. The w AlbB infection was stable at high temperatures and showed perfect maternal transmission and cytoplasmic incompatibility regardless of host background. Our results demonstrate the stability of w AlbB across host backgrounds and point to its long-term effectiveness for controlling arbovirus transmission and mosquito populations. Importance Wolbachia bacteria are being used to control the transmission of dengue and other arboviruses by mosquitoes. For Wolbachia release programs to be effective globally, Wolbachia infections must be stable across mosquito populations from different locations. In this study, we transferred Wolbachia (strain w AlbB) to Aedes aegypti mosquitoes with an Australian genotype and introduced the infection to Malaysian mosquitoes through backcrossing. We found that the phenotypic effects of Wolbachia are stable across both mosquito backgrounds. We sequenced the genome of w AlbB and found very few genetic changes despite spending over 15 years in a novel mosquito host. Our results suggest that the effects of Wolbachia infections are likely to remain stable across time and host genotype.

Immunity and nutrition are two essential modulators of individual fitness. However, while the implications of immune function and nutrition on an individual's lifespan and reproduction are well established, the interplay between feeding behaviour, infection and immune function remains poorly understood. Asking how ecological and physiological factors affect immune responses and resistance to infections is a central theme of eco‐immunology. In this study, we used the fruit fly, Drosophila melanogaster, to investigate how infection through septic injury modulates nutritional intake and how macronutrient balance affects survival to infection by the pathogenic Gram‐positive bacterium Micrococcus luteus. Our results show that infected flies maintain carbohydrate intake, but reduce protein intake, thereby shifting from a protein‐to‐carbohydrate (P:C) ratio of ~1:4 to ~1:10 relative to non‐infected and sham‐infected flies. Strikingly, the proportion of flies dying after M. luteus infection was significantly lower when flies were fed a low‐P high‐C diet, revealing that flies shift their macronutrient intake as means of nutritional self‐medication against bacterial infection. These results are likely due to the effects of the macronutrient balance on the regulation of the constitutive expression of innate immune genes, as a low‐P high‐C diet was linked to an upregulation in the expression of key antimicrobial peptides. Together, our results reveal the intricate relationship between macronutrient intake and resistance to infection and integrate the molecular cross‐talk between metabolic and immune pathways into the framework of nutritional immunology.

Mosquitoes carrying Wolbachia endosymbionts are being released in many countries for arbovirus control. The wMel strain of Wolbachia blocks Aedes-borne virus transmission and can spread throughout mosquito populations by inducing cytoplasmic incompatibility. Aedes aegypti mosquitoes carrying wMel were first released into the field in Cairns, Australia, over a decade ago, and with wider releases have resulted in the near elimination of local dengue transmission. The long-term stability of Wolbachia effects is critical for ongoing disease suppression, requiring tracking of phenotypic and genomic changes in Wolbachia infections following releases. We used a combination of field surveys, phenotypic assessments, and Wolbachia genome sequencing to show that wMel has remained stable in its effects for up to a decade in Australian Ae. aegypti populations. Phenotypic comparisons of wMel-infected and uninfected mosquitoes from near-field and long-term laboratory populations suggest limited changes in the effects of wMel on mosquito fitness. Treating mosquitoes with antibiotics used to cure the wMel infection had limited effects on fitness in the next generation, supporting the use of tetracycline for generating uninfected mosquitoes without off-target effects. wMel has a temporally stable within-host density and continues to induce complete cytoplasmic incompatibility. A comparison of wMel genomes from pre-release (2010) and nine years post-release (2020) populations show few genomic differences and little divergence between release locations, consistent with the lack of phenotypic changes. These results indicate that releases of Wolbachia-infected mosquitoes for population replacement are likely to be effective for many years, but ongoing monitoring remains important to track potential evolutionary changes.

The importance of DNA methylation in mammalian and plant systems is well established. In recent years there has been renewed interest in DNA methylation in insects. Accumulating evidence, both from mammals and insects, points towards an emerging role for DNA methylation in the regulation of phenotypic plasticity. The migratory locust (Locusta migratoria) is a model organism for the study of phenotypic plasticity. Despite this, there is little information available about the degree to which the genome is methylated in this species and genes encoding methylation machinery have not been previously identified. We therefore undertook an initial investigation to establish the presence of a functional DNA methylation system in L. migratoria. We found that the migratory locust possesses genes that putatively encode methylation machinery (DNA methyltransferases and a methyl-binding domain protein) and exhibits genomic methylation, some of which appears to be localised to repetitive regions of the genome. We have also identified a distinct group of genes within the L. migratoria genome that appear to have been historically methylated and show some possible functional differentiation. These results will facilitate more detailed research into the functional significance of DNA methylation in locusts.

Environmental DNA (eDNA) sampling-the detection of environmental cellular or extracellular DNA-has the potential to improve the sensitivity and efficiency of biodiversity surveys, enabling the coverage needed for accurate monitoring at broad spatial scales (Deiner et al., 2015;Turner et al., 2014). In aquatic environments, eDNA sampling is safer for operators (Crookes et al., 2020) and less invasive to target animals compared to traditional sampling methods, such as trapping or electrofishing (Valentini et al., 2016).

Macroinvertebrates are commonly sampled for bioassessment of freshwater ecosystems. However, current bioassessment protocols involve laborious sorting of the animals from the debris (sample matrix) and morphological identification, where species level identifications are often difficult. DNA metabarcoding has the potential to improve bioassessment by reducing the time taken to process samples and improve the accuracy and speed of macroinvertebrate species identification. In this study, we evaluated DNA metabarcoding of macroinvertebrate samples, which include macroinvertebrates and the debris collected in the sample nets, to test if bulk, unsorted samples can be used to assess macroinvertebrate diversity. First, we tested if the sample matrix prevented the detection of six target macroinvertebrate taxa when DNA metabarcoding. Second, we tested if sample storage influenced the detection of the same six target macroinvertebrates. We also explored different levels of replication at the sample, sub‐sample, and polymerase chain reaction levels and compared the overall macroinvertebrate families detected using DNA metabarcoding to those identified morphologically. We found that the presence of the sample matrix did not interfere with or inhibit the detection of the six target macroinvertebrate taxa. Furthermore, we found that the various sample storage methods did not affect target macroinvertebrate detection. The reliability of detection of the target macroinvertebrates improved as hierarchical levels of replication were combined. We found strong overlap between the detection of overall macroinvertebrate family diversity when comparing DNA metabarcoding to morphological identification. Extracting DNA from the bulk macroinvertebrate samples that included the sample matrix and using this for DNA metabarcoding could improve bioassessment by removing the need for laborious sorting of samples. Furthermore, DNA metabarcoding detection of the six target taxa was not dependent on sample storage of up to 1 year in 95% ethanol, at room temperature or after heating. DNA metabarcoding had the advantage of identifying macroinvertebrate species, but good DNA barcode libraries are needed for widespread species identifications. Further investigation should focus on including multiple samples with different macroinvertebrate composition and densities to refine and standardise bulk sample processing protocols, and on building comprehensive DNA barcode libraries for aquatic macroinvertebrates.

The green peach aphid, Myzus persicae, is globally distributed and an important pest of many economically valuable food crops, largely due to its ability to transmit plant viruses. Almost all aphids, including M. persicae, carry the obligate symbiont Buchnera aphidicola, which provides essential amino acids that aphids cannot obtain from the phloem of plants themselves. Many aphids also harbor facultative (secondary) endosymbionts, which provide benefits under specific ecological conditions. In this study, we screened for secondary endosymbionts in M. persicae, with a particular focus on Australian populations where this species is growing in status as a major agricultural pest. We compared 37 Australian M. persicae populations with other populations, including 21 field populations from China and 15 clones from the UK, France, Italy, Greece, USA, Spain, South Korea, Chile, Japan and Zimbabwe. No secondary endosymbionts were identified in M. persicae samples outside of China, despite samples covering a wide geographic range and being collected from several host plant families. We detected two secondary endosymbionts (Rickettsia, Spiroplasma) in Chinese samples, although diversity appeared lower than detected in a recent study. We also found very high clonal diversity in Chinese samples based on DNA microsatellite markers in comparison with lower clonal diversity from Australia. These patterns may indicate a higher diversity of secondary endosymbionts (and clonal diversity) in the native range of M. persicae when compared to its invasive range.