Determining Temperature Preference of Mosquitoes and Other Ectotherms.

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on their fitness, as well as other characteristics. Consequently, many such ectotherms seek out their optimal temperature range. Although temperature preferences of mosquitoes and other insects have been well studied, the traditional experimental setup is performed using a temperature gradient on an aluminum surface in a highly enclosed space. In some cases, this equipment restricts many natural behaviors, such as flying, which may be important in preference selection. The objective of this study is to observe insect preference for air temperature by using a two-chamber apparatus with sufficient room for flight. The two chambers consist of independent temperature-controlled incubators, each with a large aperture. The incubators are connected by these apertures using a short acrylic bridge. Inside the incubators are two netted cages, linked via the apertures and bridge, allowing the insects to freely fly between the different conditions. The acrylic bridge also acts as a temperature gradient between the two incubators. Due to the spacious area in the cage and easy construction, this method can be used to study any small ectotherm and/or any manipulation which may alter temperature preference including sensory organ manipulation, diet, gut flora, and endosymbiont presence at biosafety levels 1 or 2 (BSL 1 or 2). Additionally, the apparatus can be used for the study of pathogen infection using further containment (e.g., inside of a biosafety cabinet) at BSL 3.

Arsenophonus apicola sp. nov., isolated from the honeybee Apis mellifera.

The genus Arsenophonus has been traditionally considered to comprise heritable bacterial symbionts of arthropods. Recent work has reported a microbe related to the type species Arsenophonus nasoniae as infecting the honey bee, Apis mellifera. The association was unusual for members of the genus in that the microbe-host interaction arose through environmental and social exposure rather than vertical transmission. In this study, we describe the in vitro culture of ArsBeeUST, a strain of this microbe isolated from A. mellifera in the USA. The 16S rRNA sequence of the isolated strain indicates it falls within the genus Arsenophonus. Biolog analysis indicates the bacterium has a restricted range of nutrients that support growth. In vivo experiments demonstrate the strain proliferates rapidly on injection into A. mellifera hosts. We further report the closed genome sequence for the strain. The genome is 3.3 Mb and the G+C content is 37.6 mol%, which is smaller than A. nasoniae but larger than the genomes reported for non-culturable Arsenophonus symbionts. The genome is complex, with six extrachromosomal elements and 11 predicted intact phage elements, but notably less complex than A. nasoniae. Strain ArsBeeUST is clearly distinct from the type species A. nasoniae on the basis of genome sequence, with 92 % average nucleotide identity. Based on our results, we propose Arsenophonus apicola sp. nov., with the type strain ArsBeeUST (CECT 30499T=DSM113403T=LMG 32504T).

Wolbachia-Conferred Antiviral Protection Is Determined by Developmental Temperature.

Wolbachia is a maternally transmitted bacterium that is widespread in arthropods and filarial nematodes and confers strong antiviral protection in Drosophila melanogaster and other arthropods. Wolbachia-transinfected Aedes aegypti mosquitoes are currently being deployed to fight transmission of dengue and Zika viruses. However, the mechanism of antiviral protection and the factors influencing are still not fully understood. Here, we show that temperature modulates Wolbachia-conferred protection in Drosophila melanogaster. Temperature after infection directly impacts Drosophila C virus (DCV) replication and modulates Wolbachia protection. At higher temperatures, viruses proliferate more and are more lethal, while Wolbachia confers lower protection. Strikingly, host developmental temperature is a determinant of Wolbachia-conferred antiviral protection. While there is strong protection when flies develop from egg to adult at 25°C, the protection is highly reduced or abolished when flies develop at 18°C. However, Wolbachia-induced changes during development are not sufficient to limit virus-induced mortality, as Wolbachia is still required to be present in adults at the time of infection. This developmental effect is general, since it was present in different host genotypes, Wolbachia variants, and upon infection with different viruses. Overall, we show that Wolbachia-conferred antiviral protection is temperature dependent, being present or absent depending on the environmental conditions. This interaction likely impacts Wolbachia-host interactions in nature and, as a result, frequencies of host and symbionts in different climates. Dependence of Wolbachia-mediated pathogen blocking on developmental temperature could be used to dissect the mechanistic bases of protection and influence the deployment of Wolbachia to prevent transmission of arboviruses. IMPORTANCE Insects are often infected with beneficial intracellular bacteria. The bacterium Wolbachia is extremely common in insects and can protect them from pathogenic viruses. This effect is being used to prevent transmission of dengue and Zika viruses by Wolbachia-infected mosquitoes. To understand the biology of insects in the wild, we need to discover which factors affect Wolbachia-conferred antiviral protection. Here, we show that the temperature at which insects develop from eggs to adults can determine the presence or absence of antiviral protection. The environment, therefore, strongly influences this insect-bacterium interaction. Our work may help to provide insights into the mechanism of viral blocking by Wolbachia, deepen our understanding of the geographical distribution of host and symbiont, and incentivize further research on the temperature dependence of Wolbachia-conferred protection for control of mosquito-borne disease.

Thermal sensitivity of the Spiroplasma-Drosophila hydei protective symbiosis: The best of climes, the worst of climes.

The outcome of natural enemy attack in insects is commonly influenced by the presence of protective symbionts in the host. The degree to which protection functions in natural populations, however, will depend on the robustness of the phenotype and symbiosis to variation in the abiotic environment. We studied the impact of a key environmental parameter-temperature-on the efficacy of the protective effect of the symbiont Spiroplasma on its host Drosophila hydei, against attack by the parasitoid wasp Leptopilina heterotoma. In addition, we investigated the thermal sensitivity of the symbiont's vertical transmission, which may be a key determinant of the ability of the symbiont to persist. We found that vertical transmission was more robust than previously considered, with Spiroplasma being maintained at 25°C, at 18°C and with 18/15°C diurnal cycles, with rates of segregational loss only increasing at 15°C. Protection against wasp attack was ablated before symbiont transmission was lost, with the symbiont failing to rescue the fly host at 18°C. We conclude that the presence of a protective symbiosis in natural populations cannot be simply inferred from the presence of a symbiont whose protective capacity has been tested under narrow controlled conditions. More broadly, we argue that the thermal environment is likely to represent an important determinant of the evolutionary ecology of defensive symbioses in natural environments, potentially driving seasonal, latitudinal and altitudinal variation in symbiont frequency.

Infectious Diseases: Antiviral Wolbachia Limits Dengue in Malaysia.

Vector-borne viral diseases pose an urgent public health challenge, particularly in the tropics. Field releases of mosquitoes carrying bacterial symbionts that reduce vector competence are ongoing in Kuala Lumpur, Malaysia. Early results show that wAlbB Wolbachia can persist in mosquitoes in urban settings and decrease dengue incidence in humans.

Is Anopheles gambiae a Natural Host of Wolbachia?

Wolbachia (Alphaproteobacteria, Rickettsiales) is an intraovarially transmitted symbiont of insects able to exert striking phenotypes, including reproductive manipulations and pathogen blocking. These phenotypes make Wolbachia a promising tool to combat mosquito-borne diseases. Although Wolbachia is present in the majority of terrestrial arthropods, including many disease vectors, it was considered absent from Anopheles gambiae mosquitos, the main vectors of malaria in sub-Saharan Africa. In 2014, Wolbachia sequences were detected in A. gambiae samples collected in Burkina Faso. Subsequently, similar evidence came from collections all over Africa, revealing a high Wolbachia 16S rRNA sequence diversity, low abundance, and a lack of congruence between host and symbiont phylogenies. Here, we reanalyze and discuss recent evidence on the presence of Wolbachia sequences in A. gambiae. We find that although detected at increasing frequencies, the unusual properties of these Wolbachia sequences render them insufficient to diagnose natural infections in A. gambiae Future studies should focus on uncovering the origin of Wolbachia sequence variants in Anopheles and seeking sequence-independent evidence for this new symbiosis. Understanding the ecology of Anopheles mosquitos and their interactions with Wolbachia will be key in designing successful, integrative approaches to limit malaria spread. Although the prospect of using Wolbachia to fight malaria is intriguing, the newly discovered strains do not bring it closer to realization.IMPORTANCEAnopheles gambiae mosquitos are the main vectors of malaria, threatening around half of the world's population. The bacterial symbiont Wolbachia can interfere with disease transmission by other important insect vectors, but until recently, it was thought to be absent from natural A. gambiae populations. Here, we critically analyze the genomic, metagenomic, PCR, imaging, and phenotypic data presented in support of the presence of natural Wolbachia infections in A. gambiae We find that they are insufficient to diagnose Wolbachia infections and argue for the need of obtaining robust data confirming basic Wolbachia characteristics in this system. Determining the Wolbachia infection status of Anopheles is critical due to its potential to influence Anopheles population structure and Plasmodium transmission.

Within host selection for faster replicating bacterial symbionts.

Wolbachia is a widespread, intracellular symbiont of arthropods, able to induce reproductive distortions and antiviral protection in insects. Wolbachia can also be pathogenic, as is the case with wMelPop, a virulent variant of the endosymbiont of Drosophila melanogaster. An extensive genomic amplification of the 20kb region encompassing eight Wolbachia genes, called Octomom, is responsible for wMelPop virulence. The Octomom copy number in wMelPop can be highly variable between individual D. melanogaster flies, even when comparing siblings arising from a single female. Moreover, Octomom copy number can change rapidly between generations. These data suggest an intra-host variability in Octomom copy number between Wolbachia cells. Since wMelPop Wolbachia with different Octomom copy numbers grow at different rates, we hypothesized that selection could act on this intra-host variability. Here we tested if total Octomom copy number changes during the lifespan of individual Drosophila hosts, revealing selection for different Wolbachia populations. We performed a time course analysis of Octomom amplification in flies whose mothers were controlled for Octomom copy number. We show that despite the Octomom copy number being relatively stable it increases slightly throughout D. melanogaster adult life. This indicates that there is selection acting on the intra-host variation in the Octomom copy number over the lifespan of individual hosts. This within host selection for faster replicating bacterial symbionts may be in conflict with between host selection against highly pathogenic Wolbachia.

Horizontal Transmission of Intracellular Insect Symbionts via Plants.

Experimental evidence is accumulating that endosymbionts of phytophagous insects may transmit horizontally via plants. Intracellular symbionts known for manipulating insect reproduction and altering fitness (Rickettsia, Cardinium, Wolbachia, and bacterial parasite of the leafhopper Euscelidius variegatus) have been found to travel from infected insects into plants. Other insects, either of the same or different species can acquire the symbiont from the plant through feeding, and in some cases transfer it to their progeny. These reports prompt many questions regarding how intracellular insect symbionts are delivered to plants and how they affect them. Are symbionts passively transported along the insect-plant-insect path, or do they actively participate in the process? How widespread are these interactions? How does symbiont presence influence the plant? And what conditions are required for the new infection to establish in an insect? From an ecological, evolutionary, and applied perspective, this mode of horizontal transmission could have profound implications if occurring frequently enough or if new stable symbiont infections are established. Transmission of symbionts through plants likely represents an underappreciated means of infection, both in terms of symbiont epidemiology and the movement of symbionts to new host species.

Mutualism breakdown by amplification of Wolbachia genes.

Most insect species are associated with vertically transmitted endosymbionts. Because of the mode of transmission, the fitness of these symbionts is dependent on the fitness of the hosts. Therefore, these endosymbionts need to control their proliferation in order to minimize their cost for the host. The genetic bases and mechanisms of this regulation remain largely undetermined. The maternally inherited bacteria of the genus Wolbachia are the most common endosymbionts of insects, providing some of them with fitness benefits. In Drosophila melanogaster, Wolbachia wMelPop is a unique virulent variant that proliferates massively in the hosts and shortens their lifespan. The genetic bases of wMelPop virulence are unknown, and their identification would allow a better understanding of how Wolbachia levels are regulated. Here we show that amplification of a region containing eight Wolbachia genes, called Octomom, is responsible for wMelPop virulence. Using Drosophila lines selected for carrying Wolbachia with different Octomom copy numbers, we demonstrate that the number of Octomom copies determines Wolbachia titers and the strength of the lethal phenotype. Octomom amplification is unstable, and reversion of copy number to one reverts all the phenotypes. Our results provide a link between genotype and phenotype in Wolbachia and identify a genomic region regulating Wolbachia proliferation. We also prove that these bacteria can evolve rapidly. Rapid evolution by changes in gene copy number may be common in endosymbionts with a high number of mobile elements and other repeated regions. Understanding wMelPop pathogenicity and variability also allows researchers to better control and predict the outcome of releasing mosquitoes transinfected with this variant to block human vector-borne diseases. Our results show that transition from a mutualist to a pathogen may occur because of a single genomic change in the endosymbiont. This implies that there must be constant selection on endosymbionts to control their densities.

High anti-viral protection without immune upregulation after interspecies Wolbachia transfer.

Wolbachia, endosymbionts that reside naturally in up to 40-70% of all insect species, are some of the most prevalent intracellular bacteria. Both Wolbachia wAu, naturally associated with Drosophila simulans, and wMel, native to Drosophila melanogaster, have been previously described to protect their hosts against viral infections. wMel transferred to D. simulans was also shown to have a strong antiviral effect. Here we directly compare one of the most protective wMel variants and wAu in D. melanogaster in the same host genetic background. We conclude that wAu protects better against viral infections, it grows exponentially and significantly shortens the lifespan of D. melanogaster. However, there is no difference between wMel and wAu in the expression of selected antimicrobial peptides. Therefore, neither the difference in anti-viral effect nor the life-shortening could be attributed to the immune stimulation by exogenous Wolbachia. Overall, we prove that stable transinfection with a highly protective Wolbachia is not necessarily associated with general immune activation.

Wolbachia variants induce differential protection to viruses in Drosophila melanogaster: a phenotypic and phylogenomic analysis.

Wolbachia are intracellular bacterial symbionts that are able to protect various insect hosts from viral infections. This tripartite interaction was initially described in Drosophila melanogaster carrying wMel, its natural Wolbachia strain. wMel has been shown to be genetically polymorphic and there has been a recent change in variant frequencies in natural populations. We have compared the antiviral protection conferred by different wMel variants, their titres and influence on host longevity, in a genetically identical D. melanogaster host. The phenotypes cluster the variants into two groups--wMelCS-like and wMel-like. wMelCS-like variants give stronger protection against Drosophila C virus and Flock House virus, reach higher titres and often shorten the host lifespan. We have sequenced and assembled the genomes of these Wolbachia, and shown that the two phenotypic groups are two monophyletic groups. We have also analysed a virulent and over-replicating variant, wMelPop, which protects D. melanogaster even better than the closely related wMelCS. We have found that a ~21 kb region of the genome, encoding eight genes, is amplified seven times in wMelPop and may be the cause of its phenotypes. Our results indicate that the more protective wMelCS-like variants, which sometimes have a cost, were replaced by the less protective but more benign wMel-like variants. This has resulted in a recent reduction in virus resistance in D. melanogaster in natural populations worldwide. Our work helps to understand the natural variation in wMel and its evolutionary dynamics, and inform the use of Wolbachia in arthropod-borne disease control.