Luc Leblanc
University of Idaho, Department of Entomology, Plant Pathology and Nematology (EPPN), 875 Perimeter Drive MS 2329, Moscow, Idaho, USA, University of Idaho, Moscow, United States of America
M. Aftab Hossain
Insect Biotechnology Division, Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Dhaka-1349, Bangladesh, Bangladesh Atomic Energy Commission, Dhaka, Bangladesh,
Camiel Doorenweerd
Insect Biotechnology Division, Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Dhaka-1349, Bangladesh, Bangladesh Atomic Energy Commission, Dhaka, Bangladesh,
Shakil Ahmed Khan
University of Hawaii at Manoa, Department of Plant and Environmental Protection Sciences, 3050 Maile Way, Gilmore 310, Honolulu, HI 96822, USA, University of Hawaii at Manoa, Honolulu, United States of America,
Mahfuza Momen
Insect Biotechnology Division, Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Dhaka-1349, Bangladesh, Bangladesh Atomic Energy Commission, Dhaka, Bangladesh,
Michael San Jose
Insect Biotechnology Division, Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Dhaka-1349, Bangladesh, Bangladesh Atomic Energy Commission, Dhaka, Bangladesh,
Daniel Rubinoff
University of Hawaii at Manoa, Department of Plant and Environmental Protection Sciences, 3050 Maile Way, Gilmore 310, Honolulu, HI 96822, USA, University of Hawaii at Manoa, Honolulu, United States of America,
Dacini , Indian subcontinent, Pest species, Range extension, Taxonomy
Pest Management
Collecting and curation Starting in 2013, we periodically maintained a series of traps (described in separately baited with male lures plus a 10×10 mm piece of dichlorvos (DVVP) insecticide strip to kill trapped flies. Cue-lure and methyl eugenol were included as commercially available plugs (Scentry Biologicals, Billings, Montana) whereas zingerone lure, also used in the surveys since 2016, was prepared by dipping dental cotton wicks in zingerone (= vanillylacetone) (Sigma-Aldrich) melted over a hot plate and allowed to solidify in the wicks. We deployed traps at 383 sites throughout the country for periods ranging from one to 14 days, either as individual sites scattered over rural areas or as series of 11–26 sites, about 50 m apart, concentrated in selected rural areas and in 10 different protected forest areas. Sampled flies were stored in 95% ethanol in a -20 °C freezer, to preserve DNA for analysis. All flies were identified by the first three authors, using available keys. Before drying flies for double-mounting, we pinned them through the scutum with a minuten pin and soaked them in ethyl-ether for 3–12 hours to fix and preserve their natural coloration. We photographed specimens using a Nikon D7100 camera attached to an Olympus SZX10 microscope and used Helicon Focus Pro ver. 6.7.1 to merge pictures taken at a range of focal planes. To measure specimens, we used an ocular grid mounted on an Olympus SZ30 dissecting microscope. We also reared parasitoids and hyperparasitoids from readily available, heavily fly-infested snake gourd (Trichosanthes cucumerina) collected at the AERE campus (Dhaka). Infested gourds were weighed and placed on a cloth-covered small bowl (to collect excess juice from decay), over moist sawdust (as a pupation media) in a fine nylon netted cage. Pupae were separated from the sawdust using fine-meshed sieve and placed in a petri dish inside a very fine-netted plastic cage to collect emerged fruit flies and parasitoids. Morphological terms and taxonomic assignmentMorphological terminology used in the descriptions follows and assignment of species to genera follows. The genus Zeugodacus, of which a new species is described in this paper, is treated as separate from Bactrocera Macquart and Dacus Fabricius, based on recent molecular-based phylogenetic assessments . Despite recent efforts to reassign species to subgenera (e.g., Hancock and Drew 2018 a, b), the understanding of higher relationships of species within Dacini is still in state of flux, and a number of traditionally recognized subgenera and species complexes are demonstrated to be polyphyletic groups of convenience defined on the basis of highly homoplastic morphological characters and male lure relations (e.g., . For this reason, we have not attempted to include subgenera in the country’s species list. DNA extraction, PCR and sequencing Methods for DNA extraction, PCR primers and conditions, and Sanger sequencing follow those. We attempted to amplify and sequence regions of the Cytochrome C Oxidase I (COI) and Elongation Factor 1-alpha (EF1-alpha) genes. It has previously been shown that COI cannot be used to differentiate Bactrocera dorsalis from B. carambolae. However, we found that there are five diagnostic single nucleotide polymorphisms (SNP’s) that separate B. dorsalis from B. carambolae in the 762 base-pair (bp) fragment of EF1-alpha that we used for multi-marker phylogenetic studies. We therefore sequenced this segment to confirm or refute the identity of B. carambolae. For Zeugodacus madhupuri, we attempted to amplify a large section of 1540 bp of COI as well as EF1-alpha, but we only successfully amplified the COI-3P region. Amplified regions of COI-5P proved to be nuclear pseudogenes after sequencing (data not shown), and EF1-alpha did not yield any PCR product, possibly due to degradation of the template DNA. We aligned newly generated sequences with the published data of EF1-alpha or COI, respectively, from and performed maximum likelihood analyses using IQTree . We allowed IQTree to determine the substitution model via its integrated modeltest and ran a standard maximum likelihood analyses with 1000 ultrafast bootstraps and 1000 Sh-aLRT bootstraps. We consider branches with support values >95% for ultrafast bootstraps and >80% for Sh-aLRT bootstraps as well supported. Data from this study are available from the BOLDSYSTEMS Digital Repository.Estimating biodiversity We used EstimateS software to generate species accumulation curves. We estimated species diversity with the incidence-based Chao 2 algorithm, which does not include abundance in its extrapolation, thereby avoiding abundance bias in our data related to how strongly each species is attracted the lures and controlling for the predominance of a few agricultural pests in the samples. Diversity estimations were done comparing forest and rural sites, and the individual protected forest areas separately, with 100 randomizations without replacement for confidence intervals. It is understood that diversity estimates are underestimations, because they are based solely on species attracted to the male lures used in our sampling.
Zookeys. 2019; 876: 87–109. Published online 2019 Sep 25.
Journal