FAO/IAEA Agriculture and Biotechnology Laboratories

Fungal diseases in insects are common and widespread and can decimate their populations in spectacular epizootics. Virtually all insect orders are susceptible to fungal diseases, including Dipterans. Fungal pathogens such as Lagenidium, Coelomomyces and Culicinomyces are known to affect mosquito populations, and have been studied extensively. There are, however, many other fungi that infect and kill mosquitoes at the larval and/or adult stage. The discovery, in 1977, of the selective mosquito-pathogenic bacterium Bacillus thuringiensis Berliner israelensis (Bti) curtailed widespread interest in the search for other suitable biological control agents. In recent years interest in mosquito-killing fungi is reviving, mainly due to continuous and increasing levels of insecticide resistance and increasing global risk of mosquito-borne diseases. This review presents an update of published data on mosquito-pathogenic fungi and mosquito-pathogen interactions, covering 13 different fungal genera. Notwithstanding the potential of many fungi as mosquito control agents, only a handful have been commercialized and are marketed for use in abatement programs. We argue that entomopathogenic fungi, both new and existing ones with renewed/improved efficacies may contribute to an expansion of the limited arsenal of effective mosquito control tools, and that they may contribute in a significant and sustainable manner to the control of vector-borne diseases such as malaria, dengue and filariasis.

The Sterile Insect Technique (SIT) is amongst the most non-disruptive pest control methods. Unlike some other biologically-based methods it is species specific, does not release exotic agents into new environments and does not even introduce new genetic material into existing populations as the released organisms are not self-replicating. However, the SIT is only effective when integrated on an areawide basis, addressing the total population of the pest, irrespective of its distribution. There has been considerable progress in the development and integrated application of the SIT against the Mediterranean fruit fly (medfly), Ceratitis capitata, as reflected by operational programs for prevention, suppression and eradication of this pest. There is however, considerable scope for improving the efficiency of medfly SIT, an indispensable requirement for increased involvement of the private sector in any future application. One way to achieve this has been the development of genetic sexing strains, making it possible to release only sterile males. Another is improving sterile male performance through a better understanding of the sexual behavior of this insect. Unlike other insects for which the SIT has been successfully applied, medfly has a complex lek-based mating system in which the females exert the mate choice selecting among aggregated and displaying wild and sterile males. With the objective of developing a better understanding of medfly mating behavior, an FAO/IAEA Coordinated Research Project was carried out from 1994 to 1999. Some of the resulting work conducted during this period with the participation of research teams from ten countries is reported in this issue.

A total of fourteen (14) species of wild edible fruits from Burkina Faso were analyzed for their phenolic and flavonoid contents, and their antioxidant activities using the DPPH, FRAP and ABTS methods. The data obtained show that the total phenolic and total flavonoid levels were significantly higher in the acetone than in the methanol extracts.Detarium microcarpum fruit had the highest phenolic and the highest flavonoid content,followed by that of Adansonia digitata, Ziziphus mauritiana, Ximenia americana and Lannea microcarpa. Significant amounts of total phenolics were also detected in the other fruit species in the following order of decreasing levels: Tamarindus indica > Sclerocaryabirrea > Dialium guineense > Gardenia erubescens > Diospyros mespiliformis > Parkiabiglobosa > Ficus sycomorus > Vitellaria paradoxa. Detarium microcarpum fruit also showed the highest antioxidant activity using the three antioxidant assays. Fruits with high antioxidant activities were also found to possess high phenolic and flavonoid contents. There was a strong correlation between total phenolic and flavonoid levels and antioxidant activities.

A series of transposons are described which contain the gusA gene, encoding beta-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.

Abstract One of the main obstacles for a wider use of the Sterile Insect Technique (SIT) against the Mediterranean fruit fly (medfly) is the damage commercial fruit suffers due to sterile female stings. To overcome this obstacle, the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture has played a leading role in sponsoring and carrying out research to develop medfly genetic sexing strains that allow male‐only SIT releases. Recently, as a result of this continued FAO/IAEA effort, genetic sexing strains based on a temperature sensitive lethal ( tsl ) mutation have been developed at the IAEA Laboratories at Seibersdorf. Unlike previous pupal color sexing strains, these ‘second generation’ sexing strains allow female killing at an early (embryonal) stage. In addition, they are essentially stable under mass rearing conditions. This represents an important breakthrough because both of these attributes were considered indispensable for genetic sexing strains with any potential to replace conventional strains with both sexes in large scale sterile medfly production facilities. Besides the considerable savings in the costs of release and field monitoring, genetic sexing strains in field tests have shown severalfold increases in the effectiveness of the SIT as compared with the standard strains involving males and females. When releasing both males and females, sterile males are apparently not used effectively, because they use their limited sperm mostly to mate with sterile females and because they do not disperse widely in the presence of these females. When males only are released, however, they disperse much further in search of wild females and compete more intensely with wild males for wild females. As a result of the availability of usable male‐only strains, and the demonstration of their increased effectiveness, the applicability of the SIT against medfly has increased in two different ways. Highly developed commercial fruit growing regions, that previously had excluded application of SIT because of the fruit damage due to sterile female stings, are now reconsidering such free area/exclusion programs. More‐importantly, sterile male releases can now also be used for routine control purposes, rather than only for eradication programs, partially or fully replacing chemical bait‐sprays during the fruiting seasons.

Abstract Bactrocera papayae D rew & H ancock, B actrocera philippinensis D rew & H ancock, B actrocera carambolae D rew & H ancock, and B actrocera invadens D rew, T suruta & W hite are four horticultural pest tephritid fruit fly species that are highly similar, morphologically and genetically, to the destructive pest, the O riental fruit fly, B actrocera dorsalis ( H endel) ( D iptera: T ephritidae). This similarity has rendered the discovery of reliable diagnostic characters problematic, which, in view of the economic importance of these taxa and the international trade implications, has resulted in ongoing difficulties for many areas of plant protection and food security. Consequently, a major international collaborative and integrated multidisciplinary research effort was initiated in 2009 to build upon existing literature with the specific aim of resolving biological species limits among B . papayae , B . philippinensis , B . carambolae , B . invadens and B . dorsalis to overcome constraints to pest management and international trade. Bactrocera philippinensis has recently been synonymized with B . papayae as a result of this initiative and this review corroborates that finding; however, the other names remain in use. While consistent characters have been found to reliably distinguish B . carambolae from B . dorsalis , B . invadens and B . papayae , no such characters have been found to differentiate the latter three putative species. We conclude that B . carambolae is a valid species and that the remaining taxa, B . dorsalis , B . invadens and B . papayae , represent the same species. Thus, we consider B . dorsalis ( H endel) as the senior synonym of B . papayae D rew and H ancock syn.n. and B . invadens D rew, T suruta & W hite syn.n. A redescription of B . dorsalis is provided. Given the agricultural importance of B . dorsalis , this taxonomic decision will have significant global plant biosecurity implications, affecting pest management, quarantine, international trade, postharvest treatment and basic research. Throughout the paper, we emphasize the value of independent and multidisciplinary tools in delimiting species, particularly in complicated cases involving morphologically cryptic taxa.

Research on sterile mosquito technology from 1955 to the 1980s provided a substantial body of knowledge on propagation and release of sterile mosquitoes. Radiation sterilisation and chemosterilisation have been used effectively to induce dominant lethality and thereby sterilise important mosquito vectors in the laboratory. Experimental releases of chemosterilised males provided complete control of Anopheles albimanus in a small breeding population (14-15 sq km) in El Salvador. Releases of radiation sterilised males failed to control either Aedes aegypti or Anopheles quadrimaculatus in the USA. Releases of radiation-sterilised and chemosterilised male Culex quinquefasciatus in the USA and India were successful in some instances. Development of genetic sexing systems for Anopheles and improved physical separation methods for Culex have made it possible to rear and release males almost exclusively (> 99%) minimizing the release of potential vectors, the females. Factors that affected efficacy in some field programmes included reduction of competitiveness by radiation, immigration of fertilized females from outside the release zones, and inability of laboratory-bred males to perform in the wild. Despite significant progress, institutional commitments to carry the process further were generally lacking in the late 1970s and until recently. Now, with renewed interest and support for further assessment of this technology, this paper summarizes the current knowledge base, prioritizes some areas of investigation, and challenges scientists and administrators to maintain an awareness of progress, remain realistic about the interpretation of new findings, and make decisions about the sterile insect technique on the basis of informed scientific documentation. Areas recommended for priority research status include the establishment of genetic sexing mechanisms that can be transferred to other mosquito species, re-examination of radiation sterilisation, aerial release technology and mass rearing.

BACKGROUND: Malaria is one of the oldest and deadliest infectious diseases in humans. Many mathematical models of malaria have been developed during the past century, and applied to potential interventions. However, malaria remains uncontrolled and is increasing in many areas, as are vector and parasite resistance to insecticides and drugs. METHODS: This study presents a simulation model of African malaria vectors. This individual-based model incorporates current knowledge of the mechanisms underlying Anopheles population dynamics and their relations to the environment. One of its main strengths is that it is based on both biological and environmental variables. RESULTS: The model made it possible to structure existing knowledge, assembled in a comprehensive review of the literature, and also pointed out important aspects of basic Anopheles biology about which knowledge is lacking. One simulation showed several patterns similar to those seen in the field, and made it possible to examine different analyses and hypotheses for these patterns; sensitivity analyses on temperature, moisture, predation and preliminary investigations of nutrient competition were also conducted. CONCLUSIONS: Although based on some mathematical formulae and parameters, this new tool has been developed in order to be as explicit as possible, transparent in use, close to reality and amenable to direct use by field workers. It allows a better understanding of the mechanisms underlying Anopheles population dynamics in general and also a better understanding of the dynamics in specific local geographic environments. It points out many important areas for new investigations that will be critical to effective, efficient, sustainable interventions.

Abstract The use of 15 N techniques allows for the quantitative evaluation of N 2 fixation and distribution and their impact on the N balance in various soil‐plant systems. The A‐value approach was used in this investigation to assess N 2 fixed at various growth stages in fieldgrown soybean [ Glycine max (L.) Merrill] cv. Chippewa in a Typic Eutrocrepts soil. At physiological maturity (R7), the amount of N derived from fixation (Ndfa) was 102 kg/ha, equivalent to 47% of total N assimilated, while the contributions from soil (Ndfs) and 15 N‐labelled fertilizer (Ndff) accounted for 50 and 3%, respectively. Up to growth stage V6, which occupied half of the total duration of growth, Ndfa was less than 5% of N 2 fixed by physiological maturity. A rapid increase in Ndfa occurred from R1 onwards, and during the reproductive stages (R1‐R4), which spanned less than one‐third of the total duration of growth, this represented about 45% of total Ndfa. An almost equal portion of N (approximately 43%) was fixed from pod‐filling (R5) to physiological maturity (R7), a period slightly more than one‐fifth of the total duration of growth. Therefore, substantial N 2 fixation occurred during periods of active sink development and contributed more than 65% of the plant's N accrued during pod fill (R3‐R7). Nitrogen assimilated between R3 and R7 (when N 2 fixation was high) seemed to be the predominant source of N for pod development. Thus there was a greater contribution from fixed N (55%) than soil N (43%) in pods and seeds at the end of R7. After grain removal, it was estimated that the growth of cv. Chippewa in this soil led to a net soil depletion of 54 kg N/ha.

In tropical agroecosystems, limited N availability remains a major impediment to increasing yield. A 15 N‐recovery experiment was conducted in 13 diverse tropical agroecosystems. The objectives were to determine the total recovery of one single 15 N application of inorganic or organic N during three to six growing seasons and to establish whether the losses of N are governed by universal principles. Between 7 and 58% (average of 21%) of crop N uptake during the first growing season was derived from fertilizer. On average, 79% of crop N was derived from the soil. When 15 N‐labeled residues were applied, in the first growing season 4% of crop N was derived from the residues. Average recoveries of 15 N‐labeled fertilizer and residue in crops after the first growing season were 33 and 7%, respectively. Corresponding recoveries in the soil were 38 and 71%. An additional 6% of the fertilizer and 9.1% of the residue was recovered by crops during subsequent growing seasons. There were no significant differences in total 15 N recovery (average 54%) between N from fertilizer and N from residue. After five growing seasons, more residue N (40%) than fertilizer N (18%) was recovered in the soil, better sustaining the soil organic matter N content. Long‐term total recoveries of 15 N‐labeled fertilizer or residue in the crop and soil were similar. Soil N remained the primary source of N for crops. As higher rainfall and temperature tend to cause higher 15 N losses, management practices to improve N use efficiency and reduce losses in wet tropical regions will remain a challenge.

Mosquitoes, just as other insects produced for the sterile insect technique (SIT), are subjected to several unnatural processes including laboratory colonisation and large-scale factory production. After these processes, sterile male mosquitoes must perform the natural task of locating and mating with wild females. Therefore, the colonisation and production processes must preserve characters necessary for these functions. Fortunately, in contrast to natural selection which favours a suite of characteristics that improve overall fitness, colonisation and production practices for SIT strive to maximize only the few qualities that are necessary to effectively control populations. However, there is considerable uncertainty about some of the appropriate characteristics due to the lack of data. Development of biological products for other applications suggest that it is possible to identify and modify competitiveness characteristics in order to produce competitive mass produced sterile mosquitoes. This goal has been pursued - and sometimes achieved - by mosquito colonisation, production, and studies that have linked these characteristics to field performance. Parallels are drawn to studies in other insect SIT programmes and aquaculture which serve as vital technical reference points for mass-production of mosquitoes, most of whose development occurs - and characteristics of which are determined - in an aquatic environment. Poorly understood areas that require further study are numerous: diet, mass handling and genetic and physiological factors that influence mating competitiveness. Compromises in such traits due to demands to increase numbers or reduce costs, should be carefully considered in light of the desired field performance.

The success of the sterile insect technique (SIT) and other genetic strategies designed to eliminate large populations of insects relies on the efficient inundative releases of competitive, sterile males into the natural habitat of the target species. As released sterile females do not contribute to the sterility in the field population, systems for the efficient mass production and separation of males from females are needed. For vector species like mosquitoes, in which only females bite and transmit diseases, the thorough removal of females before release while leaving males competent to mate is a stringent prerequisite. Biological, genetic and transgenic approaches have been developed that permit efficient male-female separation for some species considered for SIT. However, most sex separation methods have drawbacks and many of these methods are not directly transferable to mosquitoes. Unlike genetic and transgenic systems, biological methods that rely on sexually dimorphic characters, such as size or development rate, are subject to natural variation, requiring regular adjustment and re-calibration of the sorting systems used. The yield can be improved with the optimization of rearing, but the scale of mass production places practical limits on what is achievable, resulting in a poor rearing to output ratio. High throughput separation is best achieved with scalable genetic or transgenic approaches.

Making males in a fruit fly pest The Mediterranean fruit fly or Medfly ( Ceratitis capitata ) is a global and highly destructive fruit pest. Meccariello et al. identified the master gene for male sex determination on the Y chromosome of Medfly and named it Maleness-on-the-Y ( MoY ) (see the Perspective by Makki and Meller). Flies of each sex were transformed into the other sex by genetic manipulation, and crosses of transformed files generated male and female progeny. MoY is functionally conserved in the olive fruit fly and in the invasive oriental fruit fly. This discovery has potential for insect genetic control based on mass release of sterile males and future strategies based on gene drive. Science , this issue p. 1457 ; see also p. 1380

Peste des petits ruminants (PPR) constitutes one of the major hurdles to the improvement of small-ruminant production in countries where it is endemic, directly affecting the poor, the main keepers of those species. Despite the existence of highly effective vaccines for more than 25 years, this disease remains a worrying and emerging cause of morbidity and mortality in endemic and high-risk regions of Africa, the Middle East, and Asia.

BACKGROUND: Current intra-domiciliary vector control depends on the application of residual insecticides and/or repellents. Although biological control agents have been developed against aquatic mosquito stages, none are available for adults. Following successful use of an entomopathogenic fungus against tsetse flies (Diptera: Glossinidae) we investigated the potency of this fungus as a biological control agent for adult malaria and filariasis vector mosquitoes. METHODS: In the laboratory, both sexes of Anopheles gambiae sensu stricto and Culex quinquefasciatus were passively contaminated with dry conidia of Metarhizium anisopliae. Pathogenicity of this fungus for An. gambiae was further tested for varying exposure times and different doses of oil-formulated conidia. RESULTS: Comparison of Gompertz survival curves and LT50 values for treated and untreated specimens showed that, for both species, infected mosquitoes died significantly earlier (p < 0.0001) than uninfected control groups. No differences in LT50 values were found for different exposure times (24, 48 hrs or continuous exposure) of An. gambiae to dry conidia. Exposure to oil-formulated conidia (doses ranging from 1.6 x 10(7) to 1.6 x 10(10) conidia/m2) gave LT50 values of 9.69 +/- 1.24 (lowest dose) to 5.89 +/- 0.35 days (highest dose), with infection percentages ranging from 4.4-83.7%. CONCLUSION: Our study marks the first to use an entomopathogenic fungus against adult Afrotropical disease vectors. Given its high pathogenicity for both adult Anopheles and Culex mosquitoes we recommend development of novel targeted indoor application methods for the control of endophagic host-seeking females.

BACKGROUND: In the context of the Sterile Insect Technique (SIT), radiation-induced sterility in the malaria mosquito Anopheles arabiensis Patton (Diptera: Culicidae) was studied. Male mosquitoes were exposed to gamma rays in the pupal or adult stage and dose-sterility curves were determined. METHODS: Pupae were irradiated shortly before emergence (at 22-26 hrs of age), and adults <24 hrs post emergence. Doses tested ranged between 0 and 100 Gy. The effects of irradiation on adult emergence, male survival, induced sterility and insemination capability were evaluated. Emergence and insemination data were analysed using independent t-tests against the control. Correlation analyses were performed for insemination rate and dose and insemination and fecundity. Male survival was analysed using Kaplan-Meier survival analyses. Finally, the calculated residual fertility values were inverse-normal transformed and linear regression analyses performed. RESULTS: Irradiation of pupae, for all doses tested, had no effect on adult emergence. Survival curves of males irradiated as pupae or adults were similar or even slightly higher than non-irradiated males. Overall, adults appeared to be slightly more susceptible to irradiation, although no significant differences for individual doses were observed. In the pupal stage, a significant negative correlation was found between insemination and dose, but the correlation-coefficient was associated with less than 25% of the total variation. A review of the literature indicated that An. arabiensis is more radiation resistant than other anopheline mosquitoes. CONCLUSION: The optimal dose for male insects to be released in an SIT programme depends on their level of sterility and competitiveness. The use of semi-sterilizing doses to produce more competitive insects is discussed. The most convenient developmental stage for mosquito irradiation on a mass-scale are pupae, but pupal irradiation resulted in a lower insemination rate at the highest dose compared to adult irradiation. On the basis of this study, a suitable dose range that includes semi-sterilizing doses is identified to initiate competitiveness experiments for males irradiated at both developmental stages.

The method to assess the sexual compatibility and mating performance of Mediterranean fruit fly, Ceratitis capitata (Wiedemann), mass-reared strains has been revised. Three new indices (isolation index, ISI; male relative performance index, MRPI; female relative performance index, FRPI) that look at the relative impact of both male and female population on the sexual isolation between laboratory and wild strains are described. The methodology and the indices were used in Argentina to test the SEIB 6–96 genetic sexing strain in competition with wild C. capitata population from the Patagonia region. The experiments show that the 2 populations are sexually compatible (ISI = 0.309). SEIB 6–96 sterile males compete with wild males, achieving 1 out of 4 Patagonian female mates. Using the current method as a decision tool, SEIB 6–96 sterile males are released weekly in Patagonia since September 1997. The accuracy of the 3 new indices is compared with the indices previously available. The possible application of the method and the indices in SIT operational programs world-wide is discussed.

In the medfly Ceratitis capitata, the Y chromosome is responsible for determining the male sex. We have mapped the region containing the relevant factor through the analysis of Y-autosome translocations using fluorescence in situ hybridization with two different probes. One probe, the clone pY114, contains repetitive, Y-specific DNA sequences from C. capitata, while the second clone, pDh2-H8, consists of ribosomal DNA sequences from Drosophila hydei. Clone pY114 labeled most of the long arm and pDh2-H8 hybridizes to the short arm and the centromeric region of the long arm. In 12 of the analyzed 19 Y-autosome translocation strains, adjacent-1 segregation products survive to the late pupal or even adult stage and can, therefore, be sexed. This was correlated with the length of the Y fragment still present in these aberrant individuals and allowed us to map the male-determining factor to a region of the long arm representing approximately 15% of the entire Y chromosome. No additional factors, affecting for example fertility, were detected outside the male-determining region.

Mating compatibility among different populations of the South American fruit y Anastrepha fraterculus (Wiedemann) was assessed through mating tests in pairwise combinations. Screened cages, inside a greenhouse, containing Citrus limon (L.) trees were used. Mating compatibility was determined using the index of sexual isolation. Most of the populations were noncompatible with each other and thus sexually isolated. Of these, Tucuma n (Argentina) and Piracicaba (Brazil) populations showed a lower degree of isolation, whereas the other tested combinations were highly isolated. Full mating compatibility was detected only between two Argentinean (Concordia and Tucuma n) and two Peruvian populations (La Molina and Piura La Molina). Flies were sexually active at different times: Tucuma n, Concordia, and Piracicaba populations presented an early morning peak, La Molina and Piura La Molina were active around midday, and Ibague (Colombia) were active late in the afternoon. Manipulation of light phase conditions to match the times of maximum sexual activity did not increase the compatibility between La Molina and Tucuma n. Based on these behavioral results, which conrm morphometric, genetic, and other evidence, the taxonomic revision of this cryptic species complex is warranted. One practical implication is that colonies of this pest to be used in any sterile insect technique approach should be derived from the target population or from a compatible population. Regional efforts should be initiated to determine the distribution of each subgroup and their relationship with each other in terms of compatibility.

Fungal diseases in insects are common and widespread and can decimate their populations in spectacular epizootics. Virtually all insect orders are susceptible to fungal diseases, including Dipterans. Fungal pathogens such as Lagenidium, Coelomomyces and Culicinomyces are known to affect mosquito populations, and have been studied extensively. There are, however, many other fungi that infect and kill mosquitoes at the larval and/or adult stage. The discovery, in 1977, of the selective mosquito-pathogenic bacterium Bacillus thuringiensis Berliner israelensis (Bti) curtailed widespread interest in the search for other suitable biological control agents. In recent years interest in mosquito-killing fungi is reviving, mainly due to continuous and increasing levels of insecticide resistance and increasing global risk of mosquito-borne diseases. This review presents an update of published data on mosquito-pathogenic fungi and mosquito-pathogen interactions, covering 13 different fungal genera. Notwithstanding the potential of many fungi as mosquito control agents, only a handful have been commercialized and are marketed for use in abatement programs. We argue that entomopathogenic fungi, both new and existing ones with renewed/improved efficacies may contribute to an expansion of the limited arsenal of effective mosquito control tools, and that they may contribute in a significant and sustainable manner to the control of vector-borne diseases such as malaria, dengue and filariasis.