Development of a multi-entry identification key for economically important fruit fly larvae (Diptera, Tephritidae, Dacinae).

Identification of fruit fly larvae is difficult due to the limited morphological characteristics present. However, this is the stage at which fruit flies are intercepted at ports of entry through horticultural imports. Molecular tools are useful but are time-consuming and expensive compared to morphological identifications. This project aims to use available information from the literature and our own research to build a multi-entry identification key for thirteen tephritid species and species groups that are of economic concern for the European Union. Third-instar larvae were obtained from different regions and hosts. Thirteen species or representatives of species groups were obtained, including Ceratitis, Dacus, Bactrocera and Zeugodacus spp. The cephalopharyngeal skeletons were dissected out, cleared in a 10% NaOH solution, dehydrated and mounted in Euparal on glass slides. Images of at least 20 larvae/species were captured using a compound microscope fitted with a camera. Measurements were taken of the mounted mandibles and the number of tubules and their position in the anterior spiracles in relation to the cephalic skeleton were noted. Differences between morphometric parameters were tested via ANOVA and verified using discriminant function analysis. A matrix was compiled including nine characters for which significant inter-specific differentiation was preliminarily detected. The key was converted into a mobile application by LucID.

Potential of in vivo- and in vitro-cultured entomopathogenic nematodes to infect Lobesia vanillana (Lepidoptera: Tortricidae) under laboratory conditions.

Entomopathogenic nematodes (EPNs) have been successfully applied as biological control agents against above ground and soil stages of insect pests. However, for commercial application, it is crucial to mass culture these nematodes using in vitro liquid culture technology, as it is not attainable when using susceptible insects as hosts. Lobesia vanillana (Lepidoptera: Tortricidae) is regarded a sporadic pest of wine grapes in South Africa. The in vivo- and in vitro-cultured South African EPNs, Steinernema yirgalemense and Steinernema jeffreyense (Rhabditida: Steinernematidae), were evaluated against larvae and pupae of L. vanillana in laboratory bioassays. For larvae, high mortality was observed for all treatments: In vitro-cultured S. yirgalemense (98%) performed better than S. jeffreyense (73%), while within in vivo cultures, there was no difference between nematode species (both 83%). No significant difference was detected between in vivo- and in vitro cultures of the same nematode species. The LD50 of the in vitro-cultured S. yirgalemense, was 7.33 nematodes per larva. Mortality by infection was established by dissecting L. vanillana cadavers and confirming the presence of nematodes, which was > 90% for all treatments. Within in vitro cultures, both S. yirgalemense and S. jeffreyense were able to produce a new cohort of infective juveniles from L. vanillana larvae. Pupae, however, were found to be considerably less susceptible to EPN infection. This is the first study on the use of EPNs to control L. vanillana. The relative success of in vitro-cultured EPN species in laboratory assays, without any loss in pathogenicity, is encouraging for further research and development of this technology.

Barcoding pest species in a biodiversity hot-spot: the South African polyphagous broad-nosed weevils (Coleoptera, Curculionidae, Entiminae).

Polyphagous broad nosed weevils (Curculionidae: Entiminae) constitute a large and taxonomically challenging subfamily that contains economically significant agricultural pests worldwide. South Africa is a hot-spot for biodiversity and several species of indigenous and endemic genera of Entiminae have shifted on to cultivated plants, with some being phytosanitary pests. The sporadic pest status of many species (where the species has an occasional economic impact on the agricultural industry, but is not encountered often enough that is is readily recognisable by researchers and agricultural extension workers) and the presence of pest complexes and cryptic species represent an identification challenge to non-specialists. Furthermore, no comprehensive identification tools exist to identify immature stages that may be found in crops/soil. In this paper, a curated barcoding database with 70 COI sequences from 41 species (39 Entiminae, 2 Cyclominae) is initiated, to assist with the complexity of identification of species in this group.

Characterisation of Metarhizium majus (Hypocreales: Clavicipitaceae) isolated from the Western Cape Province, South Africa.

Entomopathogenic fungi (EPF) are important soil-dwelling entomopathogens, which can be used as biological control agents against pest insects. EPF are capable of causing lethal epizootics in pest insect populations in agroecosystems. During a survey of the orchard soil at an organic farm, different EPF species were collected and identified to species level, using both morphological and molecular techniques. The EPF were trapped from soil samples taken from an apricot orchard. The traps, which were baited in the laboratory, used susceptible host insects, including the last-instar larvae of Galleria mellonella (wax moth larvae) and Tenebrio molitor (mealworm larvae). The potential pathogenicity of the local Metarhizium majus isolate was tested and verified using susceptible laboratory-reared last-instar T. molitor larvae. The identification of the M. majus isolated from South African soil was verified using both morphological and molecular techniques. The occurrence of M. majus in the South African soil environment had not previously been reported.

Comparative demography of Bactrocera dorsalis (Hendel) and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) on deciduous fruit.

Bactrocera dorsalis (Hendel) and Ceratitis capitata (Wiedemann) are highly polyphagous fruit fly species and important pests of commercial fruit in regions of the world where they are present. In South Africa, B. dorsalis is now established in the north and northeastern parts of the country. B. dorsalis is currently absent in other parts of the country including the Western Cape Province which is an important area for the production of deciduous fruit. C. capitata is widespread in South Africa and is the dominant pest of deciduous fruit. The demographic parameters of B. dorsalis and C. capitata on four deciduous fruit types Prunus persica (L.) Batsch, Prunus domestica L., Malus domestica Borkh. and Pyrus communis L. were studied to aid in predicting the potential population establishment and growth of B. dorsalis in a deciduous fruit growing environment. All deciduous fruit types tested were suitable for population persistence of both B. dorsalis and C. capitata. Development was fastest and survival highest on nectarine for both species. B. dorsalis adults generally lived longer than those of C. capitata, irrespective of the fruit types that they developed from. B. dorsalis had a higher net reproductive rate (Ro) on all deciduous fruit tested compared to C. capitata. However, the intrinsic rate of population increase was estimated to be higher for C. capitata than for B. dorsalis on all fruit types tested primarily due to C. capitata's faster generation time. Provided abiotic conditions are optimal, B. dorsalis would be able to establish and grow in deciduous fruit growing areas.

The metabolic costs of sexual signalling in the chirping katydid Plangia graminea (Serville) (Orthoptera: Tettigoniidae) are context dependent: cumulative costs add up fast.

Katydids produce acoustic signals via stridulation, which they use to attract conspecific females for mating. However, direct estimates of the metabolic costs of calling to date have produced diverse cost estimates and are limited to only a handful of insect species. Therefore, in this study, we investigated the metabolic cost of calling in an unstudied sub-Saharan katydid, Plangia graminea Using wild-caught animals, we measured katydid metabolic rate using standard flow-through respirometry while simultaneously recording the number of calls produced. Overall, the metabolic rate during calling in P. graminea males was 60% higher than the resting metabolic rate (0.443±0.056 versus 0.279±0.028 ml CO2 h-1 g-1), although this was highly variable among individuals. Although individual call costs were relatively inexpensive (ranging from 0.02 to 5.4% increase in metabolic rate per call), the individuals with cheaper calls called more often and for longer than those with expensive calls, resulting in the former group having significantly greater cumulative costs over a standard amount of time (9.5 h). However, the metabolic costs of calling are context dependent because the amount of time spent calling greatly influenced these costs in our trials. A power law function described this relationship between cumulative cost (y) and percentage increase per call (x) (y=130.21x-1.068, R2=0.858). The choice of metric employed for estimating energy costs (i.e. how costs are expressed) also affects the outcome and any interpretation of costs of sexual signalling. For example, the absolute, relative and cumulative metabolic costs of calling yielded strongly divergent estimates, and any fitness implications depend on the organism's energy budget and the potential trade-offs in allocation of resources that are made as a direct consequence of increased calling effort.

Do thermal tolerances and rapid thermal responses contribute to the invasion potential of Bactrocera dorsalis (Diptera: Tephritidae)?

Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) has shown remarkable range expansion over the past 10years and invaded several new continents including Africa. Here we report results of a detailed assessment of acute high and low temperature survival ability and the plasticity thereof, to test the hypothesis that traits of the thermal niche have contributed to the species' invasion ability. We also assess life-stage-related variation of thermal tolerances to determine potential stage-related environmental sensitivity. The temperatures at which c. 20% of the population survived of B. dorsalis were determined to be -6.5°C and 42.7°C, respectively, when using 2h exposures. Further, four life stages of B. dorsalis (egg, 3rd instar larvae, pupae and adults) were exposed to high and low discriminating temperatures to compare their thermal survival rates. The egg stage was found to be the most resistant life stage to both high and low temperatures, since 44±2.3% survived the low and 60±4.2% survived the high discriminating temperature treatments respectively. Finally, the potential for adult hardening responses to mediate tolerance of extremes was also considered using a diverse range of acute conditions (using 2h exposures to 15°C, 10°C and 5°C and 30°C, 35°C, 37°C and 39°C as hardening temperatures, and some treatments with and without recovery periods between hardening and discriminating temperature treatment). These showed that although some significant hardening responses could be detected in certain treatments (e.g. after exposure to 37°C and 39°C), the magnitude of this plasticity was generally low compared to two other wide-spread and more geographically-range-restricted con-familial species, Ceratitis capitata and C. rosa. In other words, Bactrocera dorsalis adults were unable to rapidly heat- or cold-harden to the same extent as the other Ceratitis species examined to date. These results suggest a narrower thermal niche in B. dorsalis compared to these Ceratitis species - in both basal and plastic terms - and suggests that its geographic distribution might be more restricted in consequence.

Guiding Classical Biological Control of an Invasive Mealybug Using Integrative Taxonomy.

Delottococcus aberiae De Lotto (Hemiptera: Pseudococcidae) is a mealybug of Southern African origin that has recently been introduced into Eastern Spain. It causes severe distortions on young citrus fruits and represents a growing threat to Mediterranean citrus production. So far, biological control has proven unsatisfactory due to the absence of efficient natural enemies in Spain. Hence, the management of this pest currently relies only on chemical control. The introduction of natural enemies of D. aberiae from the native area of the pest represents a sustainable and economically viable alternative to reduce the risks linked to pesticide applications. Since biological control of mealybugs has been traditionally challenged by taxonomic misidentification, an intensive survey of Delottococcus spp. and their associated parasitoids in South Africa was required as a first step towards a classical biological control programme. Combining morphological and molecular characterization (integrative taxonomy) a total of nine mealybug species were identified in this study, including three species of Delottococcus. Different populations of D. aberiae were found on wild olive trees, in citrus orchards and on plants of Chrysanthemoides monilifera, showing intra-specific divergences according to their host plants. Interestingly, the invasive mealybug populations from Spanish orchards clustered together with the population on citrus from Limpopo Province (South Africa), sharing COI haplotypes. This result pointed to an optimum location to collect natural enemies against the invasive mealybug. A total of 14 parasitoid species were recovered from Delottococcus spp. and identified to genus and species level, by integrating morphological and molecular data. A parasitoid belonging to the genus Anagyrus, collected from D. aberiae in citrus orchards in Limpopo, is proposed here as a good biological control agent to be introduced into Spain.

Food preference and foraging activity of ants: recommendations for field applications of low-toxicity baits.

Control of ants using baits of low toxicity cannot be effective without knowledge of bait distribution patterns and bait station densities, which are determined by ants' foraging activities. Furthermore, the success of toxic baits also depends upon attractiveness of bait carriers. Here, we assessed ground and vine foraging activity and food preferences for the three ant species ( Linepithema humile (Mayr) (Hymenoptera: Formicidae), Anoplolepis custodiens (F. Smith) and Crematogaster peringueyi Emery) under field conditions. We found that L. humile's vineyard foraging activity was high and that movement of ant bait by C. peringueyi and A. custodiens in the vineyard was relatively low. Consequently, more bait stations need to be dispensed for more effective control of C. peringueyi and A. custodiens than for L. humile. Different bait densities are discussed for the various ant species. Food preference trials indicated that vineyard foraging ants preferred wet bait attractants over dry ones, making liquids the most ideal carriers for baiting these ants. Linepithema humile was attracted to 25% sugar water, while C. peringueyi was attracted to both 25% sugar water and honey. Anoplolepis custodiens was attracted to tuna but was also attracted to 25% sugar water. Thus, future bait formulations should be tailor made to suit these specific food requirements if baits are to be successful in ant pest management.

Assessment of fruit fly (Diptera: Tephritidae) management practices in deciduous fruit growing areas in South Africa.

BACKGROUND: Ceratitis capitata (Wiedemann) and Ceratitis rosa Karsch are important fruit fly pests of deciduous fruit in the Western Cape Province, South Africa. The main techniques used for fruit fly control in the Western Cape are the sterile insect technique (SIT) targeting C. capitata and the bait application technique (BAT). We determined the relative success of SIT by comparing adult fly-trap catches and fruit infestation in commercial orchards between three regions under SIT and two regions under BAT in the Western Cape, from 2006 to 2008. RESULTS: Ceratitis capitata was predominant in all regions. In commercial orchards, C. capitata catches peaked towards the end of the fruiting season (March to May) and were low between July and January. During the late season, C. capitata catches were significantly higher in two of the regions under SIT. The sterile to wild male ratio in those regions was found to be mostly <1. CONCLUSION: SIT is not being properly applied in some regions. SIT should be implemented when the pest population is low. The sterile to wild fly ratios should be increased. Alternatively, BAT should be used to lower the pest population before SIT application. Control methods should be more integrated and applied area-wide.

The distribution, relative abundance, and seasonal phenology of Ceratitis capitata, Ceratitis rosa, and Ceratitis cosyra (Diptera: Tephritidae) in South Africa.

Ceratitis capitata (Wiedemann), Ceratitis rosa Karsch, and Ceratitis cosyra (Walker) are fruit fly species (Diptera: Tephritidae) of economic importance in South Africa. These pests cause direct damage to a number of commercially produced fruit and are of phytosanitary concern. A study was conducted to determine the distribution, relative abundance, and seasonal occurrence of the three species in different climatic regions of South Africa. The relative abundance and seasonal phenology of C. capitata and C. rosa were also compared between production areas and home gardens in Stellenbosch, Western Cape. Yellow bucket traps baited with Biolure were used to trap the flies over a 2-yr period in the different sampling areas. Different fruit types were sampled in Stellenbosch to determine fruit fly infestation. C. capitata was found to have a widespread distribution in South Africa, whereas C. rosa were absent from or only present in low numbers in the drier regions. C. cosyra was restricted to the North East and East coast, following a similar pattern to the distribution of marula, Sclerocarrya birrea, an important wild host. Fruit in home gardens provided a breeding ground for C. capitata and C. rosa and a source for infestation of orchards when fruit started to mature, highlighting the need for an area-wide strategy for the control of fruit flies.