Cryptic species status of Anopheles (Diptera: Culicidae) mosquitoes in Canada using a multidisciplinary approach

Many species of Anopheles mosquitoes (Diptera: Culicidae) are now recognized as species complexes whose members are often indistinguishable morphologically but identifiable based on ecological, genetic, or behavioural data. Because the members of species complexes often differ in their vector potential, accurate identification of vector species is essential for successful mosquito control. To investigate the cryptic species status of Anopheles mosquitoes in Canada, specimens were collected from across the country and examined using morphological, molecular, and ecological data. Six of the seven traditionally recognised species from Canada were collected from locations in British Columbia, Quebec, Newfoundland and Labrador, and throughout Ontario, including Anopheles barberi, An. earlei, An. freeborni, An. punctipennis, An. quadrimaculatus s.l., and An. walkeri. Variation in polymorphic traits within An. earlei, An. punctipennis, and An. quadrimaculatus s.l. were quantified and egg morphology examined using scanning electron microscopy. Morphological identification of adult and larval specimens suggested that two described cryptic species, An. perplexens and An. smaragdinus, were present in Canada. DNA sequence data were analysed for evidence of cryptic species using three molecular markers: COl, ITS2, and ITS!. Intraspecific COl variation was very low in most species «1 %), except for An. punctipennis with 2% sequence divergence between those from British Columbia (BC) and Ontario (ON), and An. walkeri with 7% sequence divergence between populations from Manitoulin Island (NO) and Long Point Provincial Park (LP). Similar patterns were also seen using ITS2 and ITS 1. Therefore, molecular data revealed the presence of two putative cryptic species within two species examined (i.e., An. walkeri and An. punctipennis), corresponding to collection location (i.e., NO vs. LP and BC vs. ON, respectively). Surprisingly, there was no molecular support for the presence of either An. perplexens or An. smaragdinus in Canada despite the morphological assessments. Ecological data from all collection sites were recorded and are available in an online database designed to manage all collection and identification data. Current bionomic information, including regional abundance, larval habitat, and species associations, was determined for each species. This multidisciplinary study of Anopheles mosquitoes is the first detailed investigation of these potential disease vectors in Canada and demonstrates the importance of an integrated approach to anopheline systematics that includes molecular data.

Coevolution of Heliconius spp. and Passiflora spp. : a phylogenetic comparison

Although a substantial amount of research has been done on all aspects ofHeliconius biology and their ecological interactions with Passiflora, there has not hitherto been a phylogenetic examination of this association for coevolution. To test the HeliconiuslPassilfora association for coevolutionary congruence, phylogenies for each group were established and compared. The phylogeny for 14 species ofHeliconiinae from Costa Rica was based on combined sequence data from rRNA ITS 2 and partial EF-1a gene regions. For the Passifloraceae, 17 host plant species were utilized to establish a phylogeny based on tRNALeucine and ITS 1/5.8S1 ITS 2 sequence data. The phylogenies for both groups were largely in agreement with current classification (for Passifloraceae) and previously established phylogenies. Associations with the large subgenera Passiflora and Decaloba correspond with the two major Advanced Radiation groups in Heliconius. Although strict congruence above subgenus level was not observed, broad scale congruence was evident. One main host shift as well as other possible explanations for lack of strict congruence are suggested.

Cell wall degrading enzymes and interaction between Trichoderma Aggressivum and Agaricus Bisporus

Agaricus bisporus is the most commonly cultivated mushroom in North America and has a great economic value. Green mould is a serious disease of A. bisporus and causes major reductions in mushroom crop production. The causative agent of green mould disease in North America was identified as Trichoderma aggressivum f. aggressivum. Variations in the disease resistance have been shown in the different commercial mushroom strains. The purpose of this study is to continue investigations of the interactions between T. aggressivum and A. bisporus during the development of green mould disease. The main focus of the research was to study the roles of cell wall degrading enzymes in green mould disease resistance and pathogenesis. First, we tried to isolate and sequence the N-acetylglucosaminidase from A. bisporus to understand the defensive mechanism of mushroom against the disease. However, the lack of genomic and proteomic information of A. bisporus limited our efforts. Next, T. aggressivum cell wall degrading enzymes that are thought to attack Agaricus and mediate the disease development were examined. The three cell wall degrading enzymes genes, encoding endochitinase (ech42), glucanase (fJ-1,3 glucanase) and protease (prb 1), were isolated and sequenced from T. aggressivum f. aggressivum. The sequence data showed significant homology with the corresponding genes from other fungi including Trichoderma species. The transcription levels of the three T. aggressivum cell wall degrading enzymes were studied during the in vitro co-cultivation with A. bisporus using R T -qPCR. The transcription levels of the three genes were significantly upregulated compared to the solitary culture levels but were upregulated to a lesser extent in co-cultivation with a resistant strain of A. bisporus than with a sensitive strain. An Agrobacterium tumefaciens transformation system was developed for T. aggressivum and was used to transform three silencing plasmids to construct three new T. aggressivum phenotypes, each with a silenced cell wall degrading enzyme. The silencing efficiency was determined by RT-qPCR during the individual in vitro cocultivation of each of the new phenotypes with A. bisporus. The results showed that the expression of the three enzymes was significantly decreased during the in vitro cocultivation when compared with the wild type. The phenotypes were co-cultivated with A. bisporus on compost with monitoring the green mould disease progression. The data indicated that prbi and ech42 genes is more important in disease progression than the p- 1,3 glucanase gene. Finally, the present study emphasises the role of the three cell wall degrading enzymes in green mould disease infection and may provide a promising tool for disease management.