A real‐time PCR toolbox for accurate identification of invasive fruit fly species

Significant plant pests such as fruit flies that travel with fresh produce between countries as eggs or larvae pose a great economic threat to the agriculture and fruit industry worldwide. Time‐limited and expensive quarantine decisions require accurate identification of such pests. Immature stages are often impossible to identify, making them a serious concern for biosecurity agencies. Use of COI barcoding PCR, often the only molecular identification resource, is time‐consuming. We assess the suitability of the COI barcoding region for real‐time PCR assays to identify four pest fruit fly species (Family: Tephritidae), in a diagnostic framework. These species, namely Mediterranean fruit fly (Ceratitis capitata), Queensland fruit fly (Bactrocera tryoni), African invader fly (Bactrocera invadens) and Island fly (Dirioxa pornia) each provide a different set of genetic species delimitation problems. We discuss the benefits and limitations of using a single‐gene TaqMan^? real‐time approach for such species. Our results indicate that COI‐based TaqMan^? real‐time PCR assays, in particular for genetically distinct species, provide an accurate, sensitive and rapid diagnostic tool.     

Preference and performance of Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae) on five commercial fruit species

Diachasmimorpha kraussii is a polyphagous endoparasitoid of dacine fruit flies. The fruit fly hosts of D. krausii, in turn, attack a wide range of fruits and vegetables. The role that fruits play in host selection behaviour of D. kraussii has not been previously investigated. This study examines fruit preference of D. kraussii through a laboratory choice‐test trial and field fruit sampling. In the laboratory trial, oviposition preference and offspring performance measures (sex ratio, developmental time, body length, hind tibial length) of D. kraussii were investigated with respect to five fruit species [Psidium guajava L. (guava), Prunis persica L. (peach), Malus domestica Borkh. (apple), Pyrus communis L. (pear) and Citrus sinensis L. (orange)], and two fruit fly species (Bactrocera jarvisi and B. tryoni). Diachasmimorpha kraussii responded to infested fruit of all fruit types in both choice and no‐choice tests, but showed stronger preference for guava and peach in the choice tests irrespective of the species of fly larvae within the fruit. The wasp did not respond to uninfested fruit. The offspring performance measures differed in a non‐consistent fashion between the fruit types, but generally wasp offspring performed better in guava, peach and orange. The offspring sex ratio, except for one fruit/fly combination (B. jarvisi in apple), was always female biased. The combined results suggest that of the five fruits tested, guava and peach are the best fruit substrates for D. krausii. Field sampling indicated a non‐random use of available, fruit fly infested fruit by D. kraussii. Fruit fly maggots within two fruit species, Plachonia careya and Terminalia cattappa, had disproportionately higher levels of D. krausii parasitism than would be expected based on the proportion of different infested fruit species sampled, or levels of fruit fly infestation within those fruit.     

Molecular Identification of Sphaceloma perseae (Avocado Scab) and its Absence in New Zealand

Avocado scab was recorded as present in New Zealand in international databases on the basis of one isolate (ICMP 10613) identified by morphological features as Sphaceloma perseae. However, sequence analysis of the rDNA internal transcribed spacer (ITS) region showed that this isolate was dissimilar to the ITS region of other Sphaceloma species, and to S. perseae. By phylogenetic analysis, isolate ICMP 10613 was identified as a species of Phaeosphaeria. To identify S. perseae reliably and quickly, specific polymerase chain reaction (PCR) primers were developed and tested. These PCR primers detected the authentic strain and another strain available from international collections, but did not detect isolate ATCC 11190, or the New Zealand isolate ICMP 10613 which were deposited as S. perseae. No other fungi commonly present in New Zealand avocado orchards were amplified by these primers, nor were three other species of Elsinoë (E. ampelina, E. fawcettii and E. pyri). By phylogenetic analysis of ITS sequence, the atypical isolate ATCC 11190 was identified as Elsinoë araliae, whereas isolate ICMP 10613 was identified as Phaeoseptoria sp. (anamorphic Phaeosphaeria). Re‐examination of the scar symptoms on New Zealand avocado fruit showed they were dissimilar to herbarium specimens of S. perseae from Florida and from Cuba. Leaf symptoms typical of this disease have not been found in New Zealand, and isolations from over 1000 scars on fruit onto selective media yielded no fungi identifiable as S. perseae. These results show that ICMP 10613 was mis‐identified as S. perseae. The record of avocado scab in New Zealand was shown to be incorrect, and there is no evidence that the causal fungus occurs in New Zealand.     

Invasion genetics of American cherry fruit fly in Europe and signals of hybridization with the European cherry fruit fly

The American cherry fruit fly is an invasive pest species in Europe, of serious concern in tart cherry production as well as for the potential to hybridize with the European cherry fruit fly, Rhagoletis cerasi L. (Diptera: Tephritidae), which might induce new pest dynamics. In the first European reports, the question arose whether only the eastern American cherry fruit fly, Rhagoletis cingulata (Loew) (Diptera: Tephritidae), is present, or also the closely related western American cherry fruit fly, Rhagoletis indifferens Curran. In this study, we investigate the species status of European populations by comparing these with populations of both American species from their native ranges, the invasion dynamics in German (first report in 1993) and Hungarian (first report in 2006) populations, and we test for signals of hybridization with the European cherry fruit fly. Although mtDNA sequence genealogy could not separate the two American species, cross‐species amplification of 14 microsatellite loci separated them with high probabilities (0.99–1.0) and provided evidence for R. cingulata in Europe. German and Hungarian R. cingulata populations differed significantly in microsatellite allele frequencies, mtDNA haplotype and wing pattern distributions, and both were genetically depauperate relative to North American populations. The diversity suggests independent founding events in Germany and Hungary. Within each country, R. cingulata displayed little or no structure in any trait, which agrees with rapid local range expansions. In cross‐species amplifications, signals of hybridization between R. cerasi and R. cingulata were found in 2% of R. cingulata individuals and in 3% of R. cerasi. All putative hybrids had R. cerasi mtDNA indicating that the original between‐species mating involved R. cerasi females and R. cingulata males.     

Multiplex real‐time PCR assays for detection of four seedborne spinach pathogens

Aims

To develop multiplex TaqMan real‐time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach.

Methods and Results

Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real‐time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real‐time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed.

Conclusions

The real‐time PCR assays were species‐specific and sensitive. Singular or multiplex real‐time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed.

Significance and Impact of the Study

The freeze‐blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real‐time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.     

Comparative demography of a specialist and generalist fruit fly: Implications for host use and pest management

Host plants used by phytophagous insects can have significant consequences on demography parameters, overall lifetime fitness and their subsequent population dynamics. Here, we conduct a comparative demographic study between the specialist Zeugodacus cucumis (French) and generalist Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) to determine whether the host plants used by these fly species play any role in their overall lifetime fitness and explains current host use patterns. These two fly species are pests within the north-eastern region of Australia and we further aimed to use complete life-history data to determine the population parameters and models that would help identify the sensitive life-history stage that could be targeted for effective field management. Eggs collected from laboratory-reared flies were inoculated into organically grown fruits of both the primary and alternate host plant cultivars of both fly species. The proportion surviving each life stage from egg through to adult and fecundity were monitored for all cohorts from the different plant cultivars. Complete stage-base life-tables for cohorts of each fly species developing from each fruit cultivar were constructed, and the key demographic parameters and population models were analysed using PopTools matrix model programme. Our results showed that the host used by each fly species had significant consequences on fly demographic parameters and hence their overall lifetime fitness. The generalist B. tryoni was able to compensate for the fitness loss experienced at the pre-adult stage by having adults with higher fecundity, but this was not the case for the specialist Z. cucumis. Stage-base population models revealed that the population growth rate of both species was highly sensitive at the adult reproductive stage, indicating that manipulating probability of survival at this life stage would effectively manage populations of these pest species. This study provides the empirical evidence of undertaking complete life history demography studies of phytophagous insects to accurately understand their lifetime fitness consequences of using a certain host, their observed host use patterns, and overall population dynamics. We suggest that any efforts to manage dacine fruit fly pest population should consider life-history consequences of host use.     

Radiation following long‐distance dispersal: the contributions of time,opportunity and diaspore morphology in Sicyos (Cucurbitaceae)

Aim  To infer the most plausible explanations for the presence of 14 species of the Neotropical cucurbit genus Sicyos on the Hawaiian Islands, two on the Galápagos Islands, two in Australia, and one in New Zealand. Location  Neotropics, the Hawaiian and Galápagos archipelagos, Australia and New Zealand. Methods  We tested long‐problematic generic boundaries in the tribe Sicyoeae and reconstructed the history of Sicyos using plastid and nuclear DNA sequences from 87 species (many with multiple accessions) representing the group’s generic and geographic diversity. Maximum likelihood and Bayesian approaches were used to infer relationships, divergence times, biogeographic history and ancestral traits. Results  Thirteen smaller genera, including Sechium, are embedded in Sicyos, which when re‐circumscribed as a monophyletic group comprises 75 species. The 14 Hawaiian species of Sicyos descended from a single ancestor that arrived c. 3 million years ago (Ma), Galápagos was reached twice at c. 4.5 and 1 Ma, the species in Australia descended from a Neotropical ancestor (c. 2 Ma), and New Zealand was reached from Australia. Time since arrival thus does not correlate with Sicyos species numbers on the two archipelagos. Main conclusions  A plausible mechanism for the four trans‐Pacific dispersal events is adherence to birds of the tiny hard fruit with retrorsely barbed spines found in those lineages that underwent long‐distance migrations. The Hawaiian clade has lost these spines, resulting in a lower dispersal ability compared with the Galápagos and Australian lineages, and perhaps favouring allopatric speciation.     

Observations of parasitoid behaviour in both no‐choice and choice tests are consistent with proposed ecological host range

The solitary larval endoparasitoid Eadya daenerys Ridenbaugh (Hymenoptera: Braconidae) is a proposed biocontrol agent of Paropsis charybdis Stål (Coleoptera: Chrysomelidae, Chrysomelinae), a pest of eucalypts in New Zealand. Eadya daenerys oviposition behaviour was examined in two assay types during host range testing, with the aim of improving ecological host range prediction. No‐choice sequential and two‐choice behavioural observations were undertaken against nine closely related species of New Zealand non‐target beetle larvae, including a native beetle, introduced weed biocontrol agents, and invasive paropsine beetles. No behavioural measure was significantly different between no‐choice and two‐choice tests. In sequential no‐choice assays the order of first presentation (target–non‐target) had no significant effect on the median number of attacks or the attack rate while on the plant. Beetle species was the most important factor. Parasitoids expressed significantly lower on‐plant attack rates against non‐targets compared to target P. charybdis larvae. The median number of attacks was always higher towards target larvae than towards non‐target larvae, except for the phylogenetically closest related non‐target Trachymela sloanei (Blackburn) (Coleoptera: Chrysomelidae, Chrysomelinae). Most non‐target larvae were disregarded upon contact, which suggests that the infrequent attack behaviour observed by two individual E. daenerys against Allocharis nr. tarsalis larvae in two‐choice tests and the frass of Chrysolina abchasica (Weise) was probably abnormal host selection behaviour. Results indicate that E. daenerys is unlikely to attack non‐target species apart from Eucalyptus‐feeding invasive paropsines (Chrysomelinae). Non‐lethal negative impacts upon less preferred non‐target larvae are possible if E. daenerys does attack them in the field; however, this is likely to be rare.     

A high‐throughput detection method for invasive fruit fly (Diptera: Tephritidae) species based on microfluidic dynamic array

Invasive species can be detrimental to a nation's ecology, economy and human health. Rapid and accurate diagnostics are critical to limit the establishment and spread of exotic organisms. The increasing rate of biological invasions relative to the taxonomic expertise available generates a demand for high‐throughput, DNA‐based diagnostics methods for identification. We designed species‐specific qPCR primer and probe combinations for 27 economically important tephritidae species in six genera (Anastrepha, Bactrocera, Carpomya, Ceratitis, Dacus and Rhagoletis) based on 935 COI DNA barcode haplotypes from 181 fruit fly species publically available in BOLD, and then tested the specificity for each primer pair and probe through qPCR of 35 of those species. We then developed a standardization reaction system for detecting the 27 target species based on a microfluidic dynamic array and also applied the method to identify unknown immature samples from port interceptions and field monitoring. This method led to a specific and simultaneous detection for all 27 species in 7.5 h, using only 0.2 μL of reaction system in each reaction chamber. The approach successfully discriminated among species within complexes that had genetic similarities of up to 98.48%, while it also identified all immature samples consistent with the subsequent results of morphological examination of adults which were reared from larvae of cohorts from the same samples. We present an accurate, rapid and high‐throughput innovative approach for detecting fruit flies of quarantine concern. This is a new method which has broad potential to be one of international standards for plant quarantine and invasive species detection.     

γ‐Octalactone,an effective oviposition stimulant of Bactrocera tryoni

Insects commonly rely on olfactory, gustatory and visual cues when deciding where to lay eggs. The olfactory cues that stimulate oviposition in the Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), are not well understood. Here, we show that two known oviposition stimulants of the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)—γ‐octalactone and benzothiazole—strongly elicit aggregation and oviposition in B. tryoni. Two other known oviposition stimulants of B. dorsalis—ethyl tiglate and 1‐octen‐3‐ol—elicit aggregation but not oviposition. Highlighting species overlap, but also differences, in oviposition stimulants, these findings have practical application for mass‐rearing in which vast numbers of flies are reared for sterile insect technique programs and may also have practical application in the development of pest management and monitoring tools.     

Genome size estimation with quantitative real‐time PCR in two Tephritidae species: Ceratitis capitata and Bactrocera oleae

The medfly Ceratitis capitata and the olive fruit fly Bactrocera oleae belong to the Tephritidae family of Diptera, a family whose members cause severe damages in agriculture worldwide. For such insect pests, the utmost concern is their population control. The sterile insect technique (SIT) has been used in the Tephritidae family with varying degrees of success. Its efficient use usually depends on the development of genetic sexing strains and the release of only male flies. However, such advances are based on modern genetic, molecular and genomic tools. The medfly is clearly the prototype of the family, since such tools have advanced considerably, which has resulted in effective SIT efforts around the world. A whole‐genome sequencing project of this insect is already underway. In contrast, similar tools in the olive fly lag behind, even though the insect is considered a promising candidate for a next SIT target. An accurate estimate of genome size provides a preliminary view of genome complexity and indicates possible difficulties in genome assembly in whole‐genome projects. Taking advantage of a quantitative real‐time PCR approach, we determined the genome size of these two species C. capitata and B. oleae as 591 Mb (CI range: 577–605 Mb) and 322 Mb (CI range: 310–334 Mb) respectively.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Fruit size preference in the New Zealand pigeon (Hemiphaga novaeseelandiae)

Abstract We investigated whether the New Zealand pigeon Hemiphaga novaeseelandiae (Columbidae) exhibits size‐based preferences for fruits. We tested the hypothesis that in small‐fruited species, pigeons would prefer larger fruits, but in larger‐fruited species, this preference would reverse as the pigeons become increasingly limited by their gape size. We collected undispersed fruits and bird‐dispersed seeds of 10 plant species, some over several sites or years (13 datasets in total). We estimated the fruit size of dispersed seeds by fitting regressions of fruit diameter to seed diameter in intact fruits. We were able to predict fruit diameter from seed diameter in 12 of the 13 populations, although the relationship was stronger in single‐seeded species than in multi‐seeded species. Seven of the 12 populations tested showed a significant difference in seed diameter among undispersed and dispersed seeds. However, our results showed no consistent pattern in fruit size preference by the New Zealand pigeon and did not support our hypothesis. The large‐bodied New Zealand pigeon is generally not gape limited and fruit size preferences appear to be independent of mean fruit size.     

Specific and Sensitive Detection of Phytophthora nicotianae by Nested PCR and Loop‐mediated Isothermal Amplification Assays

Phytophthora nicotianae is an important soilborne plant pathogen. It causes black shank in tobacco and other commercially important crop diseases. Early and accurate detection of P. nicotianae is essential for controlling these diseases. In this study, primers based on the Ras‐related protein gene (Ypt1) of P. nicotianae were tested for their specific detection of the pathogen using nested PCR and LAMP assays. For specificity testing, DNA extracts from 47 P. nicotianae isolates, 45 isolates of 16 different oomycetes and 25 isolates of other fungal species were used; no cross‐reaction with other pathogens was observed. The sensitivity assay showed that the nested PCR and LAMP assays had detection limits of 100 fg and 10 fg genomic DNA per 25‐μl reaction, respectively. Furthermore, the nested PCR and LAMP assays were used for the detection of DNA from naturally P. nicotianae‐infected tobacco tissues and soil. Our results suggest that the LAMP assay has the greatest potential for the specific detection of P. nicotianae in regions that are at risk of contracting tobacco black shank disease and that the Ypt1 gene is a novel and effective target of P. nicotianae LAMP visual detection.     

Saprophagous insect larvae,Drosophila melanogaster,profit from increased species richness in beneficial microbes

Female fruit flies, Drosophila melanogaster, lay their eggs on decaying plant material. Foraging fly larvae strongly depend on the availability of dietary microbes, such as yeasts, to reach the adult stage. In contrast, strong interference competition with filamentous fungi can cause high mortality among Drosophila larvae. Given that many insects are known for employing beneficial microbes to combat antagonistic ones, we hypothesized that fly larvae engaged in competition with the noxious mould Aspergillus nidulans benefit from the presence of dietary yeast species, especially when they are associated with increasingly species rich yeast communities (ranging from one to six yeast species per community). On a nutrient‐limited fruit substrate infested with A. nidulans, both larval survival and development time were positively affected by more diverse yeast communities. On a mould‐free fruit substrate, merely larval development but not survival was found to be affected by increasing species richness of dietary yeasts. Not only yeast diversity had an effect on D. melanogaster life‐history traits, but also the identity of the yeast combinations. These findings demonstrate the importance of the structure and diversity of microbial communities in mutualistic animal–microbe interactions.     

A virgin flight across the Tasman Sea? Satellite tracking of post-fledging movement in the Australasian Gannet Morus serrator

New technologies enable tracking of the route, duration, and destination of previously unassessed long-distance movements. Fledgling Australasian Gannets Morus serrator from breeding populations in New Zealand had been reported to fly across the Tasman Sea to Australia, with this historic knowledge derived from the recovery of banded carcasses and from observations of initial flight direction. We deployed Argos satellite devices on ten M. serrator fledglings at Cape Kidnappers Gannetry, North Island, New Zealand, across 2 years. Birds that were tracked leaving the colony initially appeared to have landed on the sea. A male bird and two female birds were tracked moving along the east coast to the south tip of New Zealand. The two females then crossed the Tasman Sea to eastern Australian coastal waters in 4 and 5 days, respectively. We suggest that, contrary to historic reports, the route via Stewart Island constitutes a realized migration path for fledglings from Cape Kidnappers, which might minimize the distance traveled across the open sea to southeastern Australia or Tasmania. Our results further imply that initial direction of flight needs not be indicative of the subsequent migration route taken by M. serrator. This highlights the importance of direct tracking technology for adequate assessment of dispersal and migration in seabirds and other highly mobile species.     

The infectivity and host range of Orgyia anartoides nucleopolyhedrovirus

The painted apple moth (PAM), Teia anartoides (Walker) (Lepidoptera: Lymantriidae) made a recent incursion into New Zealand. A nucleopolyhedrovirus (NPV), Orgyia anartoides NPV (OranNPV), originally isolated from PAM in Australia, was tested for its pathogenicity to PAM and a range of non‐target insect species found in New Zealand, to evaluate its suitability as a microbial control for this insect invader. Dosage‐mortality tests showed that OranNPV was highly pathogenic to PAM larvae; mean LT₅₀ values for third instars ranged from 17.9 to 8.1 days for doses from 10² to 10⁵ polyhedral inclusion bodies/larva, respectively. The cause of death in infected insects was confirmed as OranNPV. Molecular analysis established that OranNPV can be identified by PCR and restriction digestion, and this process complemented microscopic examination of infected larvae. No lymantriid species occur in New Zealand; however, the virus had no significant effects on species from five other lepidopteran families (Noctuidae, Tortricidae, Geometridae, Nymphalidae and Plutellidae) or on adult honeybees. Thus, all indications from this initial investigation are that OranNPV would be an important tool in the control of PAM in a future incursion of this species into New Zealand.     

Acceptance and suitability of different host stages of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) and seven other tephritid fruit fly species to Tetrastichus giffardii Silvestri (Hymenoptera: Eulophidae)

Tetrastichus giffardii Silvestri is a gregarious eulophid endoparasitoid of several tephritid fruit fly species. Host stage suitability was studied using nine age groups of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), namely, eggs less than 24 h and between 24 and 48 h old, and 1- to 7-day-old larvae. Life table studies for T. giffardii using C. capitata as host were done at 26 ± 5 °C and 55–60% RH. Egg load in relation to age of the female parasitoid was also assessed as was the effect of host deprivation on adult longevity. Host acceptance and suitability were examined with respect to eight species of tephritids. Potential hosts so tested were five Ceratitis species, the Medfly, C. capitata, the mango fruit fly, Ceratitis cosyra (Walker), the Natal fruit fly, Ceratitis rosa Karsch, Ceratitis fasciventris (Bezzi), and Ceratitis anonae Graham; two Bactrocera species, the melon fruit fly, Bactrocera cucurbitae (Coquillett) and the newly invasive Bactrocera invadens Drew, Tsuruta, and White; and one Dacus species, the lesser pumpkin fly, Dacus ciliatus Loew. No parasitoids were obtained from eggs while all larval stages were suitable though at varying degrees. Parasitism and number of progeny was related to host age in a curvilinear manner with maxima at 4- to 5-day-old larvae. By contrast, development time decreased with age of host larvae while sex ratio was not affected. The intrinsic rate of increase was 0.17 ± 0.01; gross and net reproductive rates were 64.9 ± 4.3 and 44.9 ± 3.8, respectively. Non-ovipositing females lived significantly longer than ovipositing ones. The females accepted all host species tested, but only C. capitata, D. ciliatus and, to a much lesser extent, C. cosyra were suitable. In the remaining host species, most eggs were encapsulated. In C. capitata and D. ciliatus, percent parasitism was similar, but number of progeny was lower and the sex ratio, as the proportion of females, was higher when the parasitoid was reared on D. ciliatus. Progeny per puparium were also similar for the two hosts. In the light of these results it can be concluded that T. giffardii has a narrow host range, but it attacks and successfully develops in larvae representing a wide range of ages.     

Bacteria associated with four species of Dacus (Diptera: Tephritidae) and their role in the nutrition of the larvae

Summary The bacteria associated with Dacus tryoni (Froggatt), Dacus jarvisi (Tryon), Dacus neohumeralis (Hardy) and Dacus cacuminatus (Hering) were examined. Bacteria were isolated from the surface of freshly-laid eggs, from within surface sterilised pupae, from heads and abdomens of wild and laboratory-maintained flies, and from decomposed fruits in which the wild larvae were feeding. A more diverse flora was associated with D. tryoni and D. jarvisi (15 and 14 species, respectively) than with D. neohumeralis and D. cacuminatus (9 and 6 species, respectively). Most of the bacteria belonged to the family Enterobacteriaceae and while there were similarities of bacterial associations between fly species there was no evidence of a strict symbiotic association of a particular bacterium or bacteria with each species of fly. The larvae of D. jarvisi were unable to develop normally in an artificial medium containing unhydrolysed protein and free of bacteria and on a medium containing casein and Serratia liquefaciens (isolated from the flies and shown to secrete protease) the larvae died. On the same casein medium containing Enterobacter cloacae (isolated from the flies and shown to be protease negative) the larvae developed normally. Larvae of D. tryoni and D. jarvisi were devoid of protease and cellulase activity, but contained some amylase activity. The significance of these results in terms of current hypotheses concerning symbioses between tephritids and bacteria is discussed.     

Phylogeny and biogeography of Chinese and Australasian Polystichum ferns as inferred from chloroplast trnL-F and rps4-trnS sequence data

Polystichum, one of the largest genera of ferns, occurs worldwide with the greatest diversity in southwest China and adjacent regions. Although there have been studies of Chinese Polystichum on its traditional classification, geographic distributions, and even a few on its molecular systematics, its relationships to other species outside China remain little known. Here, we investigated the phylogeny and biogeography of the Polystichum species from China and Australasia. The evolutionary relationships among 42 Polystichum species found in China (29 taxa) and Australasia (13 taxa) were inferred from phylogenetic analyses of two chloroplast DNA sequence data sets: rps4-trnS and trnL-F intergenic spacers. The divergence time between Chinese and Australasian Polystichum was estimated. The results indicated that the Australasian species comprise a monophyletic group that is nested within the Chinese diversity, and that the New Zealand species are likewise a monophyletic group nested within the Australasian species. The divergence time estimates suggested that Chinese Polystichum migrated into Australasia from around 40 Ma ago, and from there to New Zealand from about 14 Ma. The diversification of the New Zealand Polystichum species began about 10 Ma. These results indicated that Polystichum probably originated in eastern Asia and migrated into Australasia: first into Australia and then into New Zealand.