Global invasion history of the agricultural pest butterfly Pieris rapae revealed with genomics and citizen science.

The small cabbage white butterfly, Pieris rapae, is a major agricultural pest of cruciferous crops and has been introduced to every continent except South America and Antarctica as a result of human activities. In an effort to reconstruct the near-global invasion history of P. rapae, we developed a citizen science project, the "Pieris Project," and successfully amassed thousands of specimens from 32 countries worldwide. We then generated and analyzed nuclear (double-digest restriction site-associated DNA fragment procedure [ddRAD]) and mitochondrial DNA sequence data for these samples to reconstruct and compare different global invasion history scenarios. Our results bolster historical accounts of the global spread and timing of P. rapae introductions. We provide molecular evidence supporting the hypothesis that the ongoing divergence of the European and Asian subspecies of P. rapae (∼1,200 y B.P.) coincides with the diversification of brassicaceous crops and the development of human trade routes such as the Silk Route (Silk Road). The further spread of P. rapae over the last ∼160 y was facilitated by human movement and trade, resulting in an almost linear series of at least 4 founding events, with each introduced population going through a severe bottleneck and serving as the source for the next introduction. Management efforts of this agricultural pest may need to consider the current existence of multiple genetically distinct populations. Finally, the international success of the Pieris Project demonstrates the power of the public to aid scientists in collections-based research addressing important questions in invasion biology, and in ecology and evolutionary biology more broadly.

Identification of Host Blood Meals of Mosquitoes (Diptera: Culicidae) Collected at the Aripo Savannas Scientific Reserve in Trinidad, West Indies.

Surveillance for blood-fed female mosquitoes was performed between August 2015 and February 2016 at sites along the periphery of the Aripo Savannas Environmentally Reserve (ASSR) located in northeastern Trinidad, West Indies. We collected engorged female mosquitoes representing 13 species. DNA extractions from dissected abdomens were subjected to PCR amplification with three primer pairs targeting the mitochondrial cytochrome oxidase I and cytochrome b gene sequences. High-quality sequence information and host identification were obtained for 42 specimens representing eight mosquito species with at least one primer combination. A broad range of vertebrates including humans were identified, but the majority were nonhuman mammals, both domestic and wild. Domestic dogs were the most common host and may represent potential sentinel species for monitoring local enzootic arbovirus activity in Trinidad. Culex declarator Dyer and Knab and Culex nigripalpus Theobald were the most common blood-fed mosquito species comprising 79.1% of the total number identified. These species obtained blood meals from birds, nonhuman mammals, and human hosts, and therefore pose significant risks as potential bridge vectors for epizootic arbovirus transmission in the ASSR area as well as other sylvan areas in Trinidad. These data represent the first such results for Trinidad.

Multifaceted functional implications of an endogenously expressed tRNA fragment in the vector mosquito Aedes aegypti.

The mosquito Aedes aegypti is the primary vector of human arboviral diseases caused by dengue, chikungunya and Zika viruses. Many studies have shown the potential roles of small RNA molecules such as microRNA, small interfering RNA and PIWI-interacting RNA in vector mosquitoes. The function of tRNA fragments (tRF), the newly discovered class of small RNAs, in mosquitoes is not known. In this study, we show that specific tRFs are expressed in significantly differential manner between males and females of Ae. aegypti strains. Specific tRFs also show differential response during developmental transition from larvae to adults, as well as after blood feeding of adult females. The expression pattern of tRFs upon blood feeding varied depending upon if the blood contained dengue virus, and also if the females were treated with antibiotic prior to feeding to cleanse of the gut bacteria. Our findings show that a single tRF derived from the precursor sequences of a tRNA-Gly was differentially expressed between males and females, developmental transitions and also upon blood feeding by females of two laboratory strains that vary in midgut susceptibility to dengue virus infection. The multifaceted functional implications of this specific tRF suggest that biogenesis of small regulatory molecules from a tRNA can have wide ranging effects on key aspects of Ae. aegypti vector biology.

A transcriptomic survey of the impact of environmental stress on response to dengue virus in the mosquito, Aedes aegypti.

Populations of Aedes aegypti naturally exhibit variable susceptibility to dengue viruses. This natural variation can be impacted by nutritional stress resulting from larval-stage crowding, indicating the influence of environment components on the adult mosquito immune response. In particular, larval crowding was previously shown to reduce the susceptibility of adult females of a Trinidad field isolate of A. aegypti to the dengue serotype 2 (JAM1409) virus. Here, we present the first whole transcriptome study to address the impact of environmental stress on A. aegypti response to dengue virus. We examined expression profiles of adult females resulting from crowded and optimum reared larvae from the same Trinidad isolate at two critical early time points-3 and 18 hours post dengue virus infected blood meal. We exposed specimens to either a dengue or naïve blood meal, and then characterized the response in ten gene co-expression modules based on their transcriptional associations with environmental stress and time. We further analyzed the top 30 hub or master regulatory genes in each of the modules, and validated our results via qRT-PCR. These hub genes reveal which functions are critical to the mechanisms that confer dengue virus refractoriness or susceptibility to stress conditioned A. aegypti, as well as the time points at which they are most important.

Larval stress alters dengue virus susceptibility in Aedes aegypti (L.) adult females.

In addition to genetic history, environmental conditions during larval stages are critical to the development, success and phenotypic fate of the Aedes aegypti mosquito. In particular, previous studies have shown a strong genotype-by-environment component to adult mosquito body size in response to optimal vs stressed larval conditions. Here, we expand upon those results by investigating the effects of larval-stage crowding and nutritional limitation on the susceptibility of a recent field isolate of Aedes aegypti to dengue virus serotype-2. Interestingly, female mosquitoes from larvae subjected to a stressed regime exhibited significantly reduced susceptibility to disseminated dengue infection 14days post infection compared to those subjected to optimal regimes. Short term survivorship post-infected blood feeding was not significantly different. As with body size, dengue virus susceptibility of a mosquito population is determined by a combination of genetic and environmental factors and is likely maintained by balancing selection. Here, we provide evidence that under different environmental conditions, the innate immune response of field-reared mosquitoes exhibits a large range of phenotypic variability with regard to dengue virus susceptibility. Further, as with body size, our results suggest that mosquitoes reared under optimal laboratory conditions, as employed in all mosquito-pathogen studies to date, may not always be realistic proxies for natural populations.

Apoptosis-related genes control autophagy and influence DENV-2 infection in the mosquito vector, Aedes aegypti.

The mosquito Aedes aegypti is the primary urban vector for dengue virus (DENV) worldwide. Insight into interactions occurring between host and pathogen is important in understanding what factors contribute to vector competence. However, many of the molecular mechanisms for vector competence remain unknown. Our previous global transcriptional analysis suggested that differential expression of apoptotic proteins is involved in determining refractoriness vs susceptibility to DENV-2 infection in Ae. aegypti females following a DENV-infected blood meal. To determine whether DENV-refractory Ae. aegypti showed more robust apoptosis upon infection, we compared numbers of apoptotic cells from midguts of refractory and susceptible strains and observed increased numbers of apoptotic cells in only the refractory strain upon DENV-2 infection. Thereafter, we manipulated apoptosis through dsRNA interference of the initiator caspase, Aedronc. Unexpectedly, dsAedronc-treated females showed both decreased frequency of disseminated infection and decreased virus titer in infected individuals. Insect caspases have also previously been identified as regulators of the cellular recycling process known as autophagy. We observed activation of autophagy in midgut and fat body tissues following a blood meal, as well as programmed activation of several apoptosis-related genes, including the effector caspase, Casps7. To determine whether autophagy was affected by caspase knockdown, we silenced Aedronc and Casps7, and observed reduced activation of autophagy upon silencing. Our results provide evidence that apoptosis-related genes are also involved in regulating autophagy, and that Aedronc may play an important role in DENV-2 infection success in Ae. aegypti, possibly through its regulation of autophagy.