Mark K. Schutze

Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wing-shape data

BackgroundBactrocera dorsalis s.s. is a pestiferous tephritid fruit fly distributed from Pakistan to the Pacific, with the Thai/Malay peninsula its southern limit. Sister pest taxa, B. papayae and B. philippinensis, occur in the southeast Asian archipelago and the Philippines, respectively. The relationship among these species is unclear due to their high molecular and morphological similarity. This study analysed population structure of these three species within a southeast Asian biogeographical context to assess potential dispersal patterns and the validity of their current taxonomic status.ResultsGeometric morphometric results generated from 15 landmarks for wings of 169 flies revealed significant differences in wing shape between almost all sites following canonical variate analysis. For the combined data set there was a greater isolation-by-distance (IBD) effect under a ‘non-Euclidean’ scenario which used geographical distances within a biogeographical ‘Sundaland context’ (r2 = 0.772, P < 0.0001) as compared to a ‘Euclidean’ scenario for which direct geographic distances between sample sites was used (r2 = 0.217, P < 0.01). COI sequence data were obtained for 156 individuals and yielded 83 unique haplotypes with no correlation to current taxonomic designations via a minimum spanning network. beast analysis provided a root age and location of 540kya in northern Thailand, with migration of B. dorsalis s.l. into Malaysia 470kya and Sumatra 270kya. Two migration events into the Philippines are inferred. Sequence data revealed a weak but significant IBD effect under the ‘non-Euclidean’ scenario (r2 = 0.110, P < 0.05), with no historical migration evident between Taiwan and the Philippines. Results are consistent with those expected at the intra-specific level.ConclusionsBactrocera dorsalis s.s., B. papayae and B. philippinensis likely represent one species structured around the South China Sea, having migrated from northern Thailand into the southeast Asian archipelago and across into the Philippines. No migration is apparent between the Philippines and Taiwan. This information has implications for quarantine, trade and pest management.

One and the same: integrative taxonomic evidence that Bactrocera invadens (Diptera: Tephritidae) is the same species as the Oriental fruit fly Bactrocera dorsalis

The invasive fruit fly Bactrocera invadens Drew, Tsuruta & White, and the Oriental fruit fly Bactrocera dorsalis (Hendel) are highly destructive horticultural pests of global significance. Bactrocera invadens originates from the Indian subcontinent and has recently invaded all of sub‐Saharan Africa, while B. dorsalis principally occurs from the Indian subcontinent towards southern China and South‐east Asia. High morphological and genetic similarity has cast doubt over whether B. invadens is a distinct species from B. dorsalis. Addressing this issue within an integrative taxonomic framework, we sampled from across the geographic distribution of both taxa and: (i) analysed morphological variation, including those characters considered diagnostic (scutum colour, length of aedeagus, width of postsutural lateral vittae, wing size, and wing shape); (ii) sequenced four loci (ITS1, ITS2, cox1 and nad4) for phylogenetic inference; and (iii) generated a cox1 haplotype network to examine population structure. Molecular analyses included the closely related species, Bactrocera kandiensis Drew & Hancock. Scutum colour varies from red‐brown to fully black for individuals from Africa and the Indian subcontinent. All individuals east of the Indian subcontinent are black except for a few red‐brown individuals from China. The postsutural lateral vittae width of B. invadens is narrower than B. dorsalis from eastern Asia, but the variation is clinal, with subcontinent B. dorsalis populations intermediate in size. Aedeagus length, wing shape and wing size cannot discriminate between the two taxa. Phylogenetic analyses failed to resolve B. invadens from B. dorsalis, but did resolve B. kandiensis. Bactrocera dorsalis and B. invadens shared cox1 haplotypes, yet the haplotype network pattern does not reflect current taxonomy or patterns in thoracic colour. Some individuals of B. dorsalis/B. invadens possessed haplotypes more closely related to B. kandiensis than to conspecifics, suggestive of mitochondrial introgression between these species. The combined evidence fails to support the delimitation of B. dorsalis and B. invadens as separate biological species. Consequently, existing biological data for B. dorsalis may be applied to the invasive population in Africa. Our recommendation, in line with other recent publications, is that B. invadens be synonymized with B. dorsalis.

Multi‐gene phylogenetic analysis of south‐east Asian pest members of the Bactrocera dorsalis species complex (Diptera: Tephritidae) does not support current taxonomy

Bactrocera dorsalis sensu stricto, B. papayae, B. philippinensis and B. carambolae are serious pest fruit fly species of the B. dorsalis complex that predominantly occur in south‐east Asia and the Pacific. Identifying molecular diagnostics has proven problematic for these four taxa, a situation that cofounds biosecurity and quarantine efforts and which may be the result of at least some of these taxa representing the same biological species. We therefore conducted a phylogenetic study of these four species (and closely related outgroup taxa) based on the individuals collected from a wide geographic range; sequencing six loci (cox1, nad4‐3′, CAD, period, ITS1, ITS2) for approximately 20 individuals from each of 16 sample sites. Data were analysed within maximum likelihood and Bayesian phylogenetic frameworks for individual loci and concatenated data sets for which we applied multiple monophyly and species delimitation tests. Species monophyly was measured by clade support, posterior probability or bootstrap resampling for Bayesian and likelihood analyses respectively, Rosenbergs reciprocal monophyly measure, P(AB), Rodrigos (P(RD)) and the genealogical sorting index, gsi. We specifically tested whether there was phylogenetic support for the four ‘ingroup’ pest species using a data set of multiple individuals sampled from a number of populations. Based on our combined data set, Bactrocera carambolae emerges as a distinct monophyletic clade, whereas B. dorsalis s.s., B. papayae and B. philippinensis are unresolved. These data add to the growing body of evidence that B. dorsalis s.s., B. papayae and B. philippinensis are the same biological species, which poses consequences for quarantine, trade and pest management.

Evolution of lure response in tephritid fruit flies: phytochemicals as drivers of sexual selection

The males of many Bactrocera species (Diptera: Tephritidae) respond strongly and positively to a smallnumber of plant-derived chemicals (¼ male lures). Males that have imbibed the lures commonly havea mating advantage over unfed males, but no female benefits have been demonstrated for femalesmating with lure-fed males. It has been hypothesized that the strong lure response is a case of runawayselection, where males receive direct benefits and females receive indirect benefits via ‘sexy sons’,ora case of sensory bias where females have a lower threshold response to lures. To test these hypotheseswe studied the effects of lure feeding on male mating, remating and longevity; while for females that hadmated with lure-fed males we recorded mating refractoriness, fecundity, egg viability and longevity. Weused Bactrocera tryoni as our test animal and as lures the naturally occurring zingerone and chemicallyrelated, but synthetic chemical cuelure. Feeding on lures provided direct male benefits in greater matingsuccess and increased multiple mating. For the first time, we recorded direct female effects: increasedfecundity and reduced remating receptivity. Egg viability did not differ in females mated with lure-fed orunfed males. The life span of males and females exposed to lures was reduced. These results reveal direct,current-generation fitness benefits for both males and females, although the male benefits appeargreater. We discuss that while lure response is indeed likely to be a sexual selection trait, there is no needto invoke runaway selection to explain its evolution. 2013 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Mating Compatibility Among Four Pest Members of the Bactrocera dorsalis Fruit Fly Species Complex (Diptera: Tephritidae)

ABSTRACT Bactrocera dorsalis (Hendel), Bactrocera papayae Drew & Hancock, Bactrocera philippinensis Drew & Hancock, and Bactrocera carambolae Drew & Hancock are pest members within the B. dorsalis species complex of tropical fruit flies. The species status of these taxa is unclear and this confounds quarantine, pest management, and general research. Mating studies carried out under uniform experimental conditions are required as part of resolving their species limits. These four taxa were collected from the wild and established as laboratory cultures for which we subsequently determined levels of prezygotic compatibility, assessed by field cage mating trials for all pair-wise combinations. We demonstrate random mating among all pair-wise combinations involving B. dorsalis, B. papayae, and B. philippinensis. B. carambolae was relatively incompatible with each of these species as evidenced by nonrandom mating for all crosses. Reasons for incompatibility involving B. carambolae remain unclear; however, we observed differences in the location of couples in the field cage for some comparisons. Alongside other factors such as pheromone composition or other courtship signals, this may lead to reduced interspecific mating compatibility with B. carambolae. These data add to evidence that B. dorsalis, B. papayae, and B. philippinensis represent the same biological species, while B. carambolae remains sufficiently different to maintain its current taxonomic identity. This poses significant implications for this groups systematics, impacting on pest management, and international trade.

Mating Compatibility Between Bactrocera invadens and Bactrocera dorsalis (Diptera: Tephritidae)

ABSTRACT The invasive fruit fly, Bactrocera invadens Drew, Tsuruta & White, is a highly polyphagous fruit pest that occurs predominantly in Africa yet has its origins in the Indian subcontinent. It is extremely morphologically and genetically similar to the Oriental fruit fly, Bactrocera dorsalis (Hendel); as such the specific relationship between these two species is unresolved. We assessed prezygotic compatibility between B. dorsalis and B. invadens using standardized field cage mating tests, which have proven effectiveness in tephritid cryptic species studies. These tests were followed by an assessment of postzygotic compatibility by examining egg viability, larval and pupal survival, and sex ratios of offspring produced from parental and subsequent F1 crosses to examine for hybrid breakdown as predicted under a two-species hypothesis. B. dorsalis was sourced from two countries (Pakistan and China), and each population was compared with B. invadens from its type locality of Kenya. B. invadens mated randomly with B. dorsalis from both localities, and there were generally high levels of hybrid viability and survival resulting from parental and F1 crosses. Furthermore, all but one hybrid cross resulted in equal sex ratios, with the single deviation in favor of males and contrary to expectations under Haldanes rule. These data support the hypothesis that B. dorsalis and B. invadens represent the same biological species, an outcome that poses significant implications for pest management and international trade for sub-Saharan Africa.

Sexual selection in true fruit flies (Diptera: Tephritidae): transcriptome and experimental evidences for phytochemicals increasing male competitive ability

In male tephritid fruit flies of the genus Bactrocera, feeding on secondary plant compounds (sensu lato male lures = methyl eugenol, raspberry ketone and zingerone) increases male mating success. Ingested male lures alter the male pheromonal blend, normally making it more attractive to females and this is considered the primary mechanism for the enhanced mating success. However, the male lures raspberry ketone and zingerone are known, across a diverse range of other organisms, to be involved in increasing energy metabolism. If this also occurs in Bactrocera, then this may represent an additional benefit to males as courtship is metabolically expensive and lure feeding may increase a flys short‐term energy. We tested this hypothesis by performing comparative RNA‐seq analysis between zingerone‐fed and unfed males of Bactrocera tryoni. We also carried out behavioural assays with zingerone‐ and cuelure‐fed males to test whether they became more active. RNA‐seq analysis revealed, in zingerone‐fed flies, up‐regulation of 3183 genes with homologues transcripts to those known to regulate intermale aggression, pheromone synthesis, mating and accessory gland proteins, along with significant enrichment of several energy metabolic pathways and gene ontology terms. Behavioural assays show significant increases in locomotor activity, weight reduction and successful mating after mounting; all direct/indirect measures of increased activity. These results suggest that feeding on lures leads to complex physiological changes, which result in more competitive males. These results do not negate the pheromone effect, but do strongly suggest that the phytochemical‐induced sexual selection is governed by both female preference and male competitive mechanisms.

Tephritid Integrative Taxonomy: Where We Are Now, with a Focus on the Resolution of Three Tropical Fruit Fly Species Complexes

Accurate species delimitation underpins good taxonomy. Formalization of integrative taxonomy in the past decade has provided a framework for using multidisciplinary data to make species delimitation hypotheses more rigorous. We address the current state of integrative taxonomy by using as a case study an international project targeted at resolving three important tephritid species complexes: Bactrocera dorsalis complex, Anastrepha fraterculus complex, and Ceratitis FAR (C. fasciventris, C. anonae, C. rosa) complex. The integrative taxonomic approach has helped deliver significant advances in resolving these complexes: It has been used to identify some taxa as belonging to the same biological species as well as to confirm hidden cryptic diversity under a single taxonomic name. Nevertheless, the general application of integrative taxonomy has not been without issue, revealing challenges that must be considered when undertaking an integrative taxonomy project. Scrutiny of this international case study provides a unique opportunity to document lessons learned for the benefit of not only tephritid taxonomists, but also the wider taxonomic community.

Species status and population structure of the Australian Eucalyptus pest Paropsis atomaria Olivier (Coleoptera: Chrysomelidae)

1 Paropsis atomaria Olivier represents an emergent pest of Eucalyptus plantations in Queensland and New South Wales, Australia. Most prior studies on the biology and control of P. atomaria have centred on populations from Canberra in the Australian Capital Territory, but the biological relationship between beetles from Canberra and those from up to 1500 km further north are unknown.

Effects of laboratory colonization on Bactrocera dorsalis (Diptera, Tephritidae) mating behaviour: 'what a difference a year makes'.

Abstract Laboratory-reared insects are widely known to have significantly reduced genetic diversity in comparison to wild populations; however, subtle behavioural changes between laboratory-adapted and wild or ‘wildish’ (i.e., within one or very few generations of field collected material) populations are less well understood. Quantifying alterations in behaviour, particularly sexual, in laboratory-adapted insects is important for mass-reared insects for use in pest management strategies, especially those that have a sterile insect technique component. We report subtle changes in sexual behaviour between ‘wildish’ Bactrocera dorsalis flies (F1 and F2) from central and southern Thailand and the same colonies 12 months later when at six generations from wild. Mating compatibility tests were undertaken under standardised semi-natural conditions, with number of homo/heterotypic couples and mating location in field cages analysed via compatibility indices. Central and southern populations of Bactrocera dorsalis displayed positive assortative mating in the 2010 trials but mated randomly in the 2011 trials. ‘Wildish’ southern Thailand males mated significantly earlier than central Thailand males in 2010; this difference was considerably reduced in 2011, yet homotypic couples from southern Thailand still formed significantly earlier than all other couple combinations. There was no significant difference in couple location in 2010; however, couple location significantly differed among pair types in 2011 with those involving southern Thailand females occurring significantly more often on the tree relative to those with central Thailand females. Relative participation also changed with time, with more southern Thailand females forming couples relative to central Thailand females in 2010; this difference was considerably decreased by 2011. These results reveal how subtle changes in sexual behaviour, as driven by laboratory rearing conditions, may significantly influence mating behaviour between laboratory-adapted and recently colonised tephritid fruit flies over a relatively short period of time.