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PEST SURVEY CARD
APPROVED: 22 December 2020
doi:10.2903/sp.efsa.2021.EN-1998
Pest survey card on Anastrepha ludens
European Food Safety Authority (EFSA),
Jan Mertens, Martijn Schenk, Alice Delbianco, Ignazio Graziosi, Sybren Vos
Abstract
This pest survey card was prepared in the context of the EFSA mandate on plant pest surveillance (M-
2020-0114), at the request of the European Commission. Its purpose is to guide the Member States in
preparing data and information for surveys of the Mexican fruit fly Anastrepha ludens. These are
required to design statistically sound and risk-based pest surveys, in line with current international
standards. Anastrepha ludens is a clearly defined taxonomic entity, but identification of this fruit fly is
challenging as several species within the genus are morphologically similar to and may co-exist with
A. ludens. Imports of citrus fruits and fruits of several tropical hosts are subject to specific measures in
EU aiming to prevent entry of non-European Tephritidae. Nevertheless, the most likely introduction of
A. ludens would be via the transport of infested fruit followed by natural spread, which might be
facilitated by the fact that the species is considered a strong flier. The insect is currently absent from
the EU and surveys should aim at substantiating pest freedom. Adult trapping using food-based
attractants is the preferred survey method for the detection of the fly. Surveillance should preferably
be executed during the time of the season in which fruits from the main host plants are available and
abundant and the climate conditions are favourable. Anastrepha ludens is a polyphagous species. The
primary host for detection surveys in the EU would be cultivated Citrus spp., whereas other host species
should be included for delimiting surveys. The pest is expected to be able to establish in the most
southern part of the EU. Species-level identification of A. ludens requires morphological examination of
the ovipositor and especially the aculeus tip of females.
© European Food Safety Authority, 2021
Keywords: food-based attractants, Mexican fruit fly, plant pest survey, priority pest, risk-based
surveillance, trapping, Union quarantine
Requestor: European Commission
Question number: EFSA-Q-2020–00467
Correspondence: ALPHA@efsa.europa.eu
www.efsa.europa.eu/publications EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card Acknowledgements: EFSA wishes to acknowledge the Netherlands Food and Consumer Product Safety Authority (NVWA) for the support provided to this scientific output in the context of the grant GP/EFSA/ALPHA/2017/02 Lot 2 – GA02 SC 02. EFSA also wishes to thank Nikolaos Papadopoulos for reviewing this document as well as the working group members that contributed to the preparation of this output. EFSA also wishes to thank especially Giulia Mattion (trainee in the Animal and Plant Health Unit-Plant Health Team) for her support to the finalisation and publication of this survey card. Suggested citation: EFSA (European Food Safety Authority), Schenk M, Mertens J, Delbianco A, Graziosi I and Vos S, 2021. Pest survey card on Anastrepha ludens. EFSA supporting publication 2021:EN-1998. 30 pp. doi:10.2903/sp.efsa.2021.EN-1998 ISSN: 2397-8325 © European Food Safety Authority, 2021 Reproduction is authorised provided the source is acknowledged with the exception of the images listed below, for which reproduction is prohibited and permission must be sought directly from the copyright holder: Figure 1: © Jack Dykinga, USDA Agricultural Research Service, Bugwood.org; Figure 2: © EPPO Global Database; Figure 3: © Ignazio Graziosi; Figure 4: © A) Florida Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Bugwood.org; B) Enrique Antonio Hernández; C) Andrés Diaz Cervantes; D) Dina Orozco-Davila; Figure 5: © A) Jeffrey W. Lotz, Florida Department of Agriculture and Consumer Services, Bugwood.org; B) and D) Pablo Liedo; C) Andrés Diaz Cervantes; Figure 6: © Pest and Diseases Image Library, Bugwood.org; Figure 7: © A) Jeffrey W. Lotz, Florida Department of Agriculture and Consumer Services, Bugwood.org; Figure 8: © Dina Orozco-Davila; Figure 9: © A) Peggy Greb, USDA Agricultural Research Service, Bugwood.org; B) Nikolaos Papadopoulos, University of Thessaly; Figures 10–11: © Dr Vicente Hernández-Ortiz, Research scientist, INECOL and S y G editores. www.efsa.europa.eu/publications 2 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card Table of contents Abstract .........................................................................................................................................1 Introduction ...................................................................................................................................4 1. The pest and its biology ......................................................................................................4 1.1. Taxonomy ..........................................................................................................................4 1.2. EU pest regulatory status ....................................................................................................5 1.3. Pest distribution ..................................................................................................................6 1.4. Life cycle ............................................................................................................................7 1.5. Host range and main hosts ..................................................................................................8 1.6. Environmental suitability ......................................................................................................9 1.7. Spread capacity ................................................................................................................ 10 1.8. Risk factor identification .................................................................................................... 10 2. Detection, sampling and identification ................................................................................ 12 2.1. Detection .......................................................................................................................... 12 2.2. Sampling .......................................................................................................................... 16 2.3. Identification .................................................................................................................... 17 3. Key elements for survey design ......................................................................................... 20 References ................................................................................................................................... 22 General glossary for pest survey.................................................................................................... 26 www.efsa.europa.eu/publications 3 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card
Introduction
The information presented in this pest survey card was summarised from a pest report to support
ranking of EU priority pests (EFSA, 2019a), the pest categorisation of non-EU Tephritidae (EFSA PLH
Panel, 2020), the European and Mediterranean Plant Protection Organization (EPPO) Global Database
(online) and the Centre for Agriculture and Bioscience International (CABI) datasheet on
Anastrepha ludens (CABI, 2019), a risk assessment on Anastrepha species (Sequeira et al., 2001), and
other documents.
The objective of this pest survey card is to provide the relevant biological information needed to prepare
surveys for A. ludens in EU Member States (MSs) following the methodology described in EFSA (2018).
It is part of a toolkit that has been developed to assist the MSs with planning a statistically sound and
risk-based pest survey approach in line with the recommendations and guidelines provided by the
International Plant Protection Convention (IPPC) in the various International Standards for Phytosanitary
Measures (ISPM 6: FAO 2018a; ISPM 31: FAO, 2016a) and guidelines for pest surveys (FAO, 2016b).
The EFSA toolkit consists of pest-specific documents and more general documents relevant for all pests
to be surveyed:
i. Pest-specific documents:
a. The pest survey card on Anastrepha ludens 1.
ii. General documents:
a. General guidelines for statistically sound and risk‐ based surveys of plant pests (EFSA, 2020a).
b. The RiBESS+ manual2.
c. The statistical tools RiBESS+3 and SAMPELATOR4.
1. The pest and its biology
1.1. Taxonomy
Scientific name: Anastrepha ludens (Loew).
Class: Insecta, Order: Diptera, Family: Tephritidae, Genus: Anastrepha, Species: Anastrepha ludens
Synonym(s): Acrotoxa ludens Loew, Anastrepha lathana Stone, Trypeta ludens (Loew).
EPPO Code: ANSTLU.
Common name: Mexican fruit fly, Mexfly.
Anastrepha ludens is a clearly defined entity that is reported to have relatively low genetic diversity
(Ruiz-Arce et al., 2015). Identification of this fruit fly might be challenging because of its morphological
similarity with other Anastrepha spp. that may co-exist. The existence of a morphologically
indistinguishable distinct form of the species in the southernmost part of its range (Costa Rica) is
hypothesised (Jiron et al., 1988; Weems et al., 2012).
The family Tephritidae consists of a vast number of species (>5,000 described species) and includes
many species that are significant agricultural pests. The genus Anastrepha consists of over 287
described species (EFSA PLH Panel, 2020). Recently the genus has been revised to include all species
of the Toxotrypana genus as well (additional seven species). Several Anastrepha species, endemic to
the tropical and subtropical areas of the Americas, pose a major threat to commercial fruit growing
areas. According to Aluja (1994), there are seven species of economically importance: A. fraterculus,
A. grandis, A. ludens, A. obliqua, A. serpentina, A. striata and A. suspensa. The Mexican fruit fly,
A. ludens (Figure 1) is an invasive species and a major pest that threatens fruit production in tropical
1
The pest survey card will be updated in the form of a Story Map that will be available in the Plant Pests Story Maps Gallery
available online: https://efsa.maps.arcgis.com/apps/MinimalGallery/index.html?appid=f91d6e95376f4a5da206eb1815ad1489
2
https://zenodo.org/record/2541541/preview/ribess-manual.pdf
3
A tutorial video for the use of RiBESS+ is available online: https://youtu.be/qYHqrCiMxDY
4
https://shiny-efsa.openanalytics.eu/
www.efsa.europa.eu/publications 4 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card
and subtropical areas around the globe. Its larvae mainly attack plant species of the family Rutaceae
(Citrus spp.), Anacardiaceae (Mangifera indica, mango) and Myrtaceae (Psidium guajava, guava), but
also attack other fruits of economic importance.
Figure 1: Egg-lying females of the Mexican fruit fly, Anastrepha ludens on a citrus fruit (Source: Jack
Dykinga, USDA Agricultural Research Service, Bugwood.org)
Conclusions on taxonomy
The Mexican fruit fly, Anastrepha ludens, is a clearly defined taxonomic entity, but its identification
might be challenging because of its morphological similarity and co-existence with other Anastrepha
spp.
1.2. EU pest regulatory status
Anastrepha ludens is a Union quarantine pest listed in Annex II of Commission Implementing Regulation
(EU) 2019/20725. The fly is also listed as a priority pest under Commission Delegated Regulation (EU)
2019/17026 implying the obligation for annual surveys of the pest.
The import of fruits and plants for planting of several hosts of non-EU Tephritidae is currently either
prohibited or subject to special requirements (Commission Implementing Regulation (EU) 2019/2072,
Commission Implementing Regulation (EU) 2018/2019 7). Special import requirements are also in place
5
Commission Implementing Regulation (EU) 2019/2072 of 28 November 2019 establishing uniform conditions for the
implementation of Regulation (EU) 2016/2031 of the European Parliament and the Council, as regards protective measures
against pests of plants, and repealing Commission Regulation (EC) No 690/2008 and amending Commission Implementing
Regulation (EU) 2018/2019.
6
Commission Delegated Regulation (EU) 2019/1702 of 1 August 2019 supplementing Regulation (EU) 2016/2031 of the European
Parliament and of the Council by establishing the list of priority pests.
7
Commission Implementing Regulation (EU) 2018/2019 of 18 December 2018 establishing a provisional list of high risk plants,
plant products or other objects, within the meaning of Article 42 of Regulation (EU) 2016/2031 and a list of plants for which
www.efsa.europa.eu/publications 5 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card for the import of growing medium (Commission Implementing Regulation (EU) 2019/2072). The general requirements for survey of quarantine organisms in the EU territory are laid down in Regulation (EU) 2016/20318 and Commission Implementing Regulation (EU) 2020/12319. Overview of the EU regulatory status Anastrepha ludens is a Union quarantine pest and is also listed as a priority pest. Import of fruits, plants for planting of several hosts and growing medium is subject to specific measures aimed to prevent the entry of non-European Tephritidae, including A. ludens. 1.3. Pest distribution Anastrepha ludens was considered to be native to north-eastern Mexico (Baker et al., 1944), but recent genetic analysis indicates that Central America is the more likely origin for this species (Ruiz-Arce et al., 2015). The insect is widespread throughout Mexico, where it occurs in high numbers (Aluja, 1994; CABI, 2019), and is currently found in all countries in Central America (Mexico, Guatemala, Belize, El Salvador, Honduras, Nicaragua, Costa Rica and Panama) (Figure 2) (EPPO, online; Ruiz-Arce et al., 2015). Historical records of A. ludens in the Andean region of Colombia are now considered to represent A. manizaliensis (Norrbom et al., 2005). Anastrepha ludens is frequently detected in citrus production areas in the United States, including the Lower Rio Grande Valley of Texas, Arizona and California (EPPO, online; Ruiz-Arce et al., 2015). In California, frequent detection is reported in the greater San Diego area (Papadopoulos et al., 2013) followed by eradication attempts, but the recurrent nature of such outbreaks is starting debates about the true pest status (Papadopoulos et al., 2013; Carey et al., 2017; Shelly et al., 2017). Currently the pest status declared is ‘transient, under eradication’ (EPPO, online). The species has been intercepted in Florida but at a much lower frequency (Steck, 1998). No outbreaks have been reported in the EU (EUROPHYT, online-a). phytosanitary certificates are not required for introduction into the Union, within the meaning of Article 73 of that Regulation. OJ L 323 19.12.2018, pp. 1–10. 8 Regulation (EU) 2016/2031 of the European Parliament of the Council of 26 October 2016 on protective measures against pests of plants, amending Regulations (EU) No 228/2013, (EU) No 652/2014 and (EU) No 1143/2014 of the European Parliament and of the Council and repealing Council Directives 69/464/EEC, 74/647/EEC, 93/85/EEC, 98/57/EC, 2000/29/EC, 2006/91/EC and 2007/33/EC (OJ L 317 23.11.2016, p. 4). 9 Commission Implementing Regulation (EU) 2020/1231 of 27 August 2020 on the format and instructions for the annual reports on the results of the surveys and on the format of the multiannual survey programmes and the practical arrangements, respectively provided for in Articles 22 and 23 of Regulation (EU) 2016/2031 of the European Parliament and the Council. OJ L 280, 28.8.2020, pp. 1–17. www.efsa.europa.eu/publications 6 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card Figure 2: Global distribution of Anastrepha ludens. Orange countries with yellow dots indicate the presence of the pest, while purple countries/states indicate transient populations (Source: EPPO Global Database, htpps://gd.eppo.int) Conclusion on pest distribution Anastrepha ludens is present in Central America (transient in southwestern USA) and currently absent from the EU. Surveys in the EU would therefore be aimed at substantiating pest freedom. 1.4. Life cycle Anastrepha ludens is a multivoltine, non-diapausing species that, depending on the climate conditions and host availability, can complete many generations per year (Figure 3). The basic life cycle among Anastrepha species is highly similar (Aluja, 1994). Soon after attaining adulthood, adults forage on sugar- and protein-rich food and reach sexual maturity in approximately two weeks (Christenson and Foote, 1960; Carey et al., 2005). Similar to other fruit flies, females of Anastrepha spp. lay eggs in the peel or the flesh (mesocarp) of ripening or ripe fruits (Christenson and Foote, 1960) and only when fruits are still hanging on the trees. The long ovipositor allows females to avoid laying eggs in the rind of the fruit and even to deposit eggs close to the seeds of the fruit. Depending on the host fruit, and the ripening stage, eggs are laid in clutches that vary from one to 40 eggs (Birke, 1995; Leyva et al., 1991; Aluja et al., 2000; Díaz‐Fleischer and Aluja, 2003). The average life span of females has been estimated at ~50 days and the maximum may exceed ~100 days (Carey et al., 2005). Lifetime fecundity rates are high as well and they have been estimated at 1500 eggs per female (Liedo et al., 1992; Carey et al., 2005). Oviposition stings may become visible in certain fruit and resin-like exudate can be found near the oviposition site in some of them. Eggs usually hatch within 4–6 days (Leyva et al., 1991), emerging larvae feed for 15–32 days when the temperature is 25°C (Christenson and Foote, 1960) and complete three larval instars. The developmental time depends on the host species and it is faster in peach (~10 days) compared with mango (~22 days), and the slowest in citrus fruits (~28 days) (Leyva et al., 1991). The larvae tunnel through the flesh of the fruits while feeding, therefore causing deterioration of the fruit. Mature larvae emerge from the fruit and drop to the ground to pupate in the soil (Christenson and Foote, 1960; Carey et al., 2005). Occasionally, larvae pupate inside the host fruit (Aluja, 1994). Pupation lasts approximately 13 to 19 days (Christenson and Foote, 1960; Leyva et al., 1991; Carey et al., 2005). www.efsa.europa.eu/publications 7 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card Figure 3: Life cycle of the Mexican fruit fly Anastrepha ludens (Courtesy of Ignazio Graziosi) Conclusion on life cycle Anastrepha ludens can complete multiple generations per year. Females lay eggs in the peel or deeper in the pulp of ripening/ripe fruits. The larvae feed in the flesh of the fruit causing deterioration that often is intensified by bacteria and fungi infections. Mature larvae leave the fruits (some may pupate inside the fruit) and drop to the ground to pupate in the soil. 1.5. Host range and main hosts Anastrepha ludens is a polyphagous species. The natural host of A. ludens is the fruit species Casimiroa (C. edulis, C. greggii, C. tetrameria) belonging to the family Rutaceae (Baker et al., 1944; Jiron et al., 1988; Pecina-Quintero et al., 2020), which can be found in the wild and in cultivation. However, Citrus species (Rutaceae) and Mangifera indica (mango, Anacardiaceae) are considered to be the main commercial hosts (CABI, 2019). EFSA (2019a) has compiled a list of host plants for A. ludens by merging information from the CABI Crop Protection Compendium, the EPPO Global Database (online) and the list of species reported by USDA Aphis (USDA, 2016). The host range information is likely to be incomplete, particularly for wild hosts. Citrus species are the main hosts of economic importance in the EU. The host range of A. ludens covers a great variety of Citrus, including C. sinensis (sweet orange), C. paradisi (grapefruit), C. reticulata (mandarin), C. aurantifolia (key lime), C. aurantium (sour orange), C. maxima (pomelo), and C. medica (citron) (Sequeira et al., 2001; CABI, 2019; EFSA 2019a). Occasional hosts that are grown as fruit crops in the Mediterranean region include Diospyros kaki (persimmon), Persea americana (avocado), www.efsa.europa.eu/publications 8 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card Pyrus communis (pear), Prunus persica (peach and nectarine), Annona squamosa (custard apple), and Punica granatum (pomegranate). In the absence of preferred hosts, occasional hosts may serve as breeding hosts and sustain populations of A. ludens. Malus domestica (apple) is reported to be suitable for development of A. ludens under laboratory conditions (Aluja et al., 2014) but this observation awaits confirmation under natural conditions. The host range of A. ludens includes several species that are primarily tropical (Sequeira et al., 2001; EFSA, 2019a). Mangifera indica (mango) is the main tropical host. Note that mango production is limited in the EU and takes place particularly in the province of Malaga in southern Spain. Besides mango, the fly also attacks various other tropical hosts, including Acca sellowiana (feijoa), Anacardium occidentale (cashew tree), several Annona spp. (including A. cherimola, A. liebmanniana, A. muricata and A. reticulata), Carica papaya (papaya), Casimiroa edulis (White sapote), Casimiroa tetramaeria (yellow sapote), Coffea arabica (coffea), Inga spp. (Ice Cream Bean), Mammea americana (mamey), Passiflora edulis (passion fruit), Psidium cattleianum (strawberry guava), Psidium guajava (guava), Sargentia greggii (yellow chapote), Spondias purpurea (jocote) and Syzygium jambos (malabar plum). These tropical hosts are mainly of relevance when determining the risk locations (import, packaging stations), while the hosts that are cultivated in the EU are relevant both for selecting risk locations and for the selection of hosts to be surveyed. Based on the above-mentioned information, the EPPO Global Database (online) and CABI (2019), the main host plants for targeted surveillance in the EU should be citrus species. These are also one of the main hosts of Bactrocera dorsalis, meaning that the surveillance of both pests can be combined, considering different trapping systems (EFSA, 2019b). Citrus is cultivated on a large scale in the EU, with the main production areas located in Spain. Spain is responsible for ~60% of the EU citrus fruit production, followed by Italy (~25%), and Greece (10%). Conclusion on host range and main hosts Anastrepha ludens is a polyphagous species. The primary host for detection surveys in the EU would be Citrus species, while other host species should be included for surveillance in case of delimiting surveys. 1.6. Environmental suitability Anastrepha ludens is a (sub)tropical species. Cold stress is likely to be the most limiting climate variable for the establishment of this species in the EU. However, given that data on cold tolerance and the overwintering potential of this species are limited, it is very difficult to infer the potential establishment of this species in the southern regions of the EU. Prolonged exposure (11 days) to temperatures of 1.1°C resulted in larval mortality of 99% (Hallman, 1999). Anastrepha ludens is not known to have any adaptations for overwintering survival, such as diapause (EFSA, 2019a). Regarding the situation in the continental part of the United States, based on host availability, generation potential and temperature requirements, Sequeira et al. (2001) indicate that the likelihood for establishment is high in Florida and in the southern parts of Texas, Georgia, South Carolina, Louisiana, Arizona and California. EFSA (2019a) used a degree day model to map potential A. ludens distribution in the EU and concluded that only the southern part the EU is considered to be suitable. This area covers central and southern Spain, central and southern Portugal, Madeira, the Azores, southern Italy, Malta, southern Greece and Cyprus. According to Aluja (1994), relative humidity is another important abiotic factor that determines development and persistence of A. ludens. Pupal desiccation in dried soil appears to be a major mortality factor (Aluja, 1994) and the optimal humidity for survival is quite high (70–100%). The degree day model used by EFSA (2019a) does not take into account relative humidity; large parts of the potential distribution area may be suboptimal for establishment of A. ludens. However, irrigation in commercial orchards and backyards should be also considered. Establishment of A. ludens is also conditional on the presence of suitable host plants. In the southern parts of the Mediterranean area, the availability of citrus fruit from September to June, and subsequent presence of several other host species from May to September, ensures that a continuous supply of fresh fruit is available. In cooler areas of the EU, host fruit availability and winter temperatures would www.efsa.europa.eu/publications 9 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card be limiting factors for establishment. Conclusion on environmental suitability Anastrepha ludens is expected to be able to establish in the southern parts of the EU, particularly in the citrus-growing areas. 1.7. Spread capacity Natural spread Anastrepha ludens is considered a strong flier (CABI, 2019). Adults can actively move many kilometres in search for hosts for oviposition. The pest is reported to fly as far as 135 km from breeding sites in Mexico to arrive at citrus groves in southern Texas (McAllister and Clore, 1941; Christenson and Foote, 1960). In release/recapture experiments adults have been trapped up to 36 km from the release site (Shaw et al., 1967). Aluja (1994) emphasises the effect of wind on displacement of the flies. Nonetheless, it is expected that flies remain in the area where they emerged to lay eggs for the next generation as long as sufficient food sources and oviposition sites are available in the nearby surroundings. Based on expert knowledge elicitation, EFSA (2019a) estimated that the maximum distance of natural spread of A. ludens is 9.4 km in one year (with a 95% uncertainty range of 2.4– 24.6 km). This scenario considers the limited need for dispersal in the Mediterranean area given the high likelihood of encountering suitable hosts in close vicinity. Given the tropical origin of the species, severe bottlenecks in population density are expected because of the Mediterranean winter, reducing spread capacity at the beginning of the season. Human-assisted spread The most likely pathway for human-assisted spread of A. ludens is through transport of infested fruit (imports of fruit commodities or fruit in passenger luggage). Because the development of eggs and larvae takes place inside the fruit, it is challenging to intercept infested fruits. The likelihood of importing adults can be considered negligible. According to the EUROPHYT and TRACES databases (online), Anastrepha species and particularly A. ludens were regularly intercepted upon entry into the EU in consignments of infested fruit from countries where the pest is present. In most cases, introduction of immature life stages of A. ludens through infested fruit may hardly result in outbreaks, because – providing the eggs or larvae survive the transport – larvae still need to conclude their development, successfully pupate and then survive to reach the adult stage. Adult females subsequently need to locate both a suitable mate and a suitable host plant bearing ripe or ripening fruits in order to oviposit and breed the next generation of flies. Pupae may also be transported in soil or other growing medium with host plants. However, the risk for entry of pupae of A. ludens in growing medium is mitigated by the general import requirements under Commission Implementing Regulation (EU) 2019/2072. Conclusion on spread capacity The maximum distance of natural spread expected to be covered in one year by A. ludens is approximately 9.4 km (with a 95% uncertainty range of 1 km to 34 km). Introduction of the pest into new areas is most likely to occur through movement of infested fruits. 1.8. Risk factor identification Identification of risk factors and their relative risk estimation are essential for performing risk-based surveys. A risk factor is a biotic or abiotic factor that increases the probability of infestation by the pest in the area of interest. The risk factors that are relevant for surveillance need to be characterised by their relative risk (should have more than one level of risk for the target population) and the proportion of the overall target population on which they apply. The identification of risk factors needs to be tailored to the situation of each Member State. This section presents examples of risk factors for A. ludens and is not necessarily exhaustive (Table 1). www.efsa.europa.eu/publications 10 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card
For the identification of risk areas, it is first necessary to identify the activities that could contribute to
introduction or spread of A. ludens. These activities should then be connected to specific locations.
Around these locations, risk areas can be defined, knowing that their size depends on the spread
capacity of the target pest and the availability of host plants around these locations.
Example 1: Entry points, packing and sorting stations of host fruits imported from regions
where the pest is present
Although the import of the main host fruits and the most relevant occasional hosts (except for guava)
is subject to special import requirements, the risk of introduction of A. ludens via import of infested
fruits cannot be excluded as shown by the regular interceptions upon entry into the EU. Entry points
(e.g. seaports, airports) of host fruits, the packing and sorting stations, and processing industries that
handle fruits originating from areas where A. ludens is present would be locations with a higher
probability of detecting the pest. This is particularly true when the imported fruits originate from areas
where A. ludens is regularly reported. Risk areas are then represented by areas where host plants are
present in the vicinity of those risk locations.
The actual risk of a packing and sorting station depends on the storage facilities and the waste disposal
procedures. If fruits are stored and handled on site while being cooled, and waste is disposed of in
closed containers, the risk for introduction at that site will not be high. Given that A. ludens is a strong
flier with a high capacity for natural spread (Section 1.7), the risk area should be of considerable size.
An area of that size is likely to contain suitable host plants. However, because host fruit abundance in
the risk area follows major seasonal fluctuations, the relative risk of an area depends on the timing of
the import, packing or processing activities. For example, when these activities take place in the winter
months, the risk of establishment is lower compared with when these activities take place during the
summer months. Similarly, when the environmental conditions near an import, packing or processing
facilities do not permit establishment because of the cool climate conditions, it is not useful to consider
such sites as risk locations unless the aim is to assess the risk of entry.
Example 2: Urban areas
Since the import of infested fruits from areas where A. ludens is present constitutes the most likely
pathway for introduction, the households that buy such host fruits and fresh markets where these fruits
are sold would be locations with a higher probability of detecting the pest. When host fruits are heavily
infested by A. ludens larvae they are more likely to be disposed of in containers or compost heaps.
Hence, waste collection centres should also be considered as risk locations. The ‘density’ of host fruit
consumption (and therefore disposal) would be higher in urban areas compared with less densely
populated areas. The preferred survey sites would then be locations with e.g. cultivated citrus trees in
or near to urban areas. In addition, hosts grown in backyards (as well as abandoned groves and
orchards) may represent a higher risk for establishment because they are usually not properly managed
or regularly harvested, therefore prolonging the period in which fruits are present. Surveillance may
include other fruit tree species that are hosts and are grown in backyards in urban areas as well.
Table 1: Examples of a risk activity and corresponding risk locations relevant for surveillance of
Anastrepha ludens
Risk activity Risk locations Risk areas
Entry points, packing and sorting stations and
Areas surrounding risk locations, where
Import of fruits of processing industries where host fruits are
host plants are present
hosts plants from handled
countries where Households, fresh markets and waste
Areas surrounding risk locations, where
the pest is present collection centres where host fruits are being
host plants are present
consumed, sold and disposed of
www.efsa.europa.eu/publications 11 EFSA Supporting publication 2021:EN-1998Anastrepha ludens survey card 2. Detection, sampling and identification 2.1. Detection Trapping adults using traps baited with food lures is the recommended method to detect A. ludens, as no male-specific attractants are available. Examining fruits for oviposition sites is possible, but the success rate will vary depending on the fruit type and the severity of infestation. When examinations of fruit are already performed to detect other pests, it might be worthwhile to include A. ludens in that respective surveillance protocol. 2.1.1. Visual examination Visual examination should focus on the detection of damage in potentially infested fruits. This would result in detection of immature stages. Although identification of third stage larvae is often possible, it requires a high magnification and expertise and is therefore not recommended. A detailed description of all immature stages of A. ludens can be found in Carroll and Wharton (1989) and Weems et al. (2012). A simplified description of all stages of the pest is given below for completeness. Pest Adapted from Carroll and Wharton (1989): the eggs are 1.37–1.60 mm long, white, spindle-shaped, broad anteriorly, tapering posteriorly. The micropyle is slightly to one side of the apex of the anterior pole. There is faint reticulation near the micropyle consisting primarily of irregular pentagons and hexagons, these becoming very faint and elongated in posterior portion of egg. The third instar larva measures 5.8–11.1 mm in length. It has a white colour and its cylindrical, elongated, slightly recurved shape is typical of Tephritidae larvae. The larva has two well developed mouth hooks. The larvae of A. ludens can be separated from those of A. fraterculus and A. obliqua by having more than 12 buccal carinae (compared with the usually eight or nine in the other two species) and by having the caudal papillae, above and below the posterior spiracles, arranged in two lines, rather than a single line. Adults (Figures 4 and 5) are 7–11 mm long, and the wings measure 7–9 mm. The colour of the body is primarily yellow to orange brown with usually darker brown setae. Several areas of the thorax have a lighter, often contrasting, shade of yellow. The abdomen lacks darker markings. Wing vein M is strongly curved anteriorly, a distinguishing feature of the Anastrepha genus as in most Tephritidae, this vein meets the costa without a curve (Figure 6). Many Anastrepha species have a characteristic wing patterns and this is often sufficient to separate them from other tephritid genera. The wing pattern of A. ludens is yellowish in colour but not unique within the genus; among others, it resembles A. distincta which naturally occurs in the same area. The female terminalia, more specifically the aculeus, allow for species- level identification. The aculeus of A. ludens is very long (3.35–5.76 mm) relative to its body size. Therefore, female specimens of A. ludens are unlikely to be confused with other Anastrepha species within its native range (CABI, 2019). However, outside its native range, other species with long aculeae exist (e.g. Anastrepha manizaliensis and Anastrepha schultzi) and a close examination of the aculea’s dimensions and features is required (Norrbom et al., 2005). www.efsa.europa.eu/publications 12 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card Figure 4: Adults: (A) and (B) adult female of Anastrepha ludens; (C) and (D) ovipositing females of A. ludens. The long ovipositor facilitates sexing of adults (Source: A) Florida Division of Plant Industry, Florida Department of Agriculture and Consumer Services, Bugwood.org; B) Enrique Antonio Hernández; C) Andrés Diaz Cervantes; D) Dina Orozco-Davila) Figure 5: Adults: (A) adult male of Anastrepha ludens; (B) marked male; (C) male of A. ludens performing sexual signalling; (D) A. ludens mating pair on the underside of a leaf (Source: A) Jeffrey W. Lotz, Florida Department of Agriculture and Consumer Services, Bugwood.org; B) and D) Pablo Liedo; C) Andrés Diaz Cervantes) www.efsa.europa.eu/publications 13 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card A B Figure 6: Wings: (A) wing of Anastrepha ludens with a strongly curved vein M (arrow); (B) wing of B. dorsalis where the vein meets the costa without such a curve (Source: Pest and Diseases Image Library, Bugwood.org) Symptoms Fruits can be examined to detect oviposition signs that indicate possible infestation of A. ludens. When the female lays eggs, this leaves a puncture mark (i.e. oviposition sting) on the skin of the attacked fruits (Figure 7). However, depending on the fruit type, it might be difficult to spot the oviposition site. In that case, the symptoms only become visible once substantial damage has occurred inside the fruit. In some sugary fruits, a resin-like exudate could be found near the oviposition site, indicating a possible infestation at an early stage (CABI, 2019). Eggs might be found inside the fruit at the location of the oviposition puncture, but such punctures would initially be difficult to detect unless examined under a microscope and by an expert entomologist. Larvae (Figure 8) can be detected when opening the fruit, especially once they have reached the advanced third instars. Figure 7: Oviposition sign (black arrow) of Anastrepha ludens on citrus fruit and an adult male (Source: Jeffrey W. Lotz, Florida Department of Agriculture and Consumer Services, Bugwood.org) www.efsa.europa.eu/publications 14 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card Figure 8: Anastrepha ludens larvae in citrus and mango fruit (Source: Dina Orozco-Davila) 2.1.2. Trapping As with other regulated Tephritidae, trapping of adult flies is the recommended and most commonly applied survey strategy (e.g. EFSA, 2019b; EFSA, 2020b). Guidance on trapping Anastrepha fruit flies is given in appendix 1 of ISPM 26 (FAO, 2018b). In contrast with the trapping of Bactrocera species, no specific male lures are available and trapping of adult flies should be carried out by using generic food lures. Traps should be placed in the upper 2/3 of the tree canopy, approximately at 1.5 to 2 m heights depending on the tree size, and on the eastern side assuring that leaves do not block trap entrance (FAO, 2018b). The trap density depends on the relative risk of the survey area. USDA (2015) suggests 1-2 trap/square mile in low risk areas, 2-5 in medium risk areas and 5-10 in high risk areas. Food-based attractants can be used for trapping both male and female flies and can be composed of synthetic volatile components of hydrolysed protein substances or the hydrolysed protein substances themselves. ISPM 26 (FAO, 2018b) recommends the use of liquid protein attractants or two-component synthetic food attractant (ammonium acetate and putrescine). Torula yeast is often used in the USA as liquid protein (Thomas et al., 2001). McPhail-type traps are used as a reservoir for these attractants (Figure 9). The original McPhail trap is made of glass; but plastic versions of the original design (e.g. the MultiLure trap) are commonly used. The use of these systems is expected to result in female-biased captures (Hall et al., 2005). Food-based attractants are generic by nature, and traps tend to capture a wide range of other non-target tephritids and non-tephritid fruit flies next to the target species. Therefore, food-based attractants will also capture fruit flies other than Anastrepha species. Hall et al. (2005) and Thomas et al. (2001; 2008) tested a variety of combinations of lures and traps. Overall, they found that: (a) a combination of ammonium acetate and putrescine was equally or more attractive than traps baited with torula yeast, (b) ammonium acetate outperformed ammonium bicarbonate, and (c) the two-component attractant of ammonium acetate and putrescine outperformed more complex formulations. In addition, the use of propylene glycol (antifreeze) as killing agent increased captures significantly and improved the preservation of specimens (Thomas et al. 2001; Hall et al., 2005). The synthetic food attractant captured fewer non-target insects and required less frequent replacements (Thomas et al., 2001). This saves considerable time in trap maintenance and requires less time to sort the captured insects. However, liquid protein baits outperformed the synthetic food attractant for Anastrepha obliqua (Thomas et al., 2008). The attractants should be carefully selected depending on the range of target species. The use of food-based attractants can be beneficial when targeting multiple species at the same time but could also result in overlooking the target species when non-target species (e.g. Ceratitis capitata) dominate captures. In general, the range of attraction of food-based attractants is lower than the range of attraction of male lures. On the other hand, food-based attractants may be more sensitive for early detection of low adult populations of fruit flies (Papadopoulos et al., 2001). Samples from the traps should be collected once or twice per week and taken to the laboratory for further confirmation, and lures should be replaced at regular intervals. www.efsa.europa.eu/publications 15 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card Figure 9: Examples of McPhail-type traps: (A) in this case the trap is used to attract Medfly (Ceratitis capitata); (B) glass McPhail used in Mexico for Anastrepha ludens (Source: A) Peggy Greb, USDA Agricultural Research Service, Bugwood.org; B) Nikolaos Papadopoulos, University of Thessaly) 2.1.3. Timing of the surveys Because it is a polyphagous pest, A. ludens can exploit different host fruits throughout the year when climate conditions are favourable (see Section 1.5). The exact timing of the surveys will depend both on the targeted host plant and the environmental conditions in the survey area. This will differ between Member States. In northern Mexico, captures of A. ludens adults follow a rather bimodal seasonal pattern with one major peak in the spring and a minor one in autumn (Thomas, 2003). However, in Texas, adult captures peak in the spring (Mangan et al., 2011). The above information on the phenology of A. ludens may not be directly related to European conditions. The seasonal patterns of fruiting and harvesting of major hosts of A. ludens should be considered to determine which is the optimum time for trapping the pest in the EU. Trapping should be conducted during fruiting and ripening seasons, when adult flies are active. Presence of ripe or ripening fruit, together with optimum climate conditions for the activity of the fruit flies in an area, greatly increases the probability of trapping adult flies, so the timing should be adjusted to specific host species and local conditions. For citrus fruits, the ripening calendar of the different varieties in a production area could be used for deploying the traps in the orchards. For example, in Spain for citrus production in the Valencian autonomous community, a ripening calendar is presented in the pest survey card for Phyllosticta citricarpa (EFSA, 2020c). Conclusions for detection methods The recommended method for the detection of Anastrepha ludens is using traps with food-based attractants to capture adult flies. The preferred seasonal window for surveillance is during the months in which abundant ripening fruits are available on the main host plants and temperature conditions are favourable for adult activity. 2.2. Sampling Any potentially infested fruit should be collected, e.g. in sealed bags, and taken to the laboratory for further confirmation. www.efsa.europa.eu/publications 16 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card
Live larvae should be collected from infested fruits and then reared to adulthood for confirmation based
on adult morphology. This is required to accurately identify A. ludens or other Anastrepha species. The
collection of living larvae is particularly important for an initial finding, but would not be needed when
dealing with known outbreaks.
If the larvae are dead or need to be preserved, they should be placed in boiling water for a few seconds.
Once they become immobile, they can either be transferred to 70% ethanol (for morphological
identification at the genus level) or to 95% ethanol (for molecular tests to support identification).
Samples from traps should be collected once or twice per week and taken to the laboratory for further
confirmation. Adult specimens should be also preserved in 95% ethanol for possible future molecular
analysis.
Conclusions for sampling
Live larvae, specimens stored on ethanol and trapped insects should be taken to the laboratory for
further identification and confirmation.
2.3. Identification
Unambiguous identification of Anastrepha ludens to species level requires morphological examination
of the ovipositor in adult females. Detailed descriptions of the egg and larval stages exist (Carroll and
Wharton, 1989), as well as a key for identification of several Anastrepha larvae (Steck et al., 1990).
Regardless of the availability of such descriptions, species-level identification based on immature stages
cannot be carried out with full certainty and should be avoided. Identifying A. ludens based on which
fruit species is infested is also impossible because of its polyphagous nature and because larvae of other
fruit flies may also infest the same fruit species. This implies that larvae should be reared to the adult
stage to allow confirmation of their identity. According to the IPPC diagnostic protocol on the genus
Anastrepha (FAO, 2016c):
‘The fruits are placed in cages covered with cloth or fine mesh and that have a sterile pupation
medium (e.g. damp vermiculite, sand or sawdust) at the bottom. Once the larvae emerge from
the fruit, they will move to the substratum for pupation. It is recommended to incubate each fruit
separately. Each sample must be observed and pupae gathered daily. The pupae are placed in
containers with the pupation medium, and the containers are covered with a tight lid that enables
proper ventilation. Once the adults emerge, they must be kept alive for 48–72 h to ensure that
the tegument and wings acquire the rigidity and characteristic colouration of the species. The
adults are then killed and preserved by placing them in 70% ethanol … or they are killed with
ethyl acetate or another agent and then mounted on pins. For female flies, immediately after
killing them (before they harden) it is useful to gently squeeze the apical part of the preabdomen
with forceps, then squeeze the base and apex of the oviscape to expose the aculeus tip (so that
it does not need to be dissected later).’
Keys for the morphological identification of Tephritidae are available (Brown et al., 2010). Identification
of the Anastrepha genus is based on wing venation characteristics (Norrbom, 2010). To observe the
relevant features for genus identification, a ×10 magnification is sufficient. Species-level identification
is not as straightforward as several species within the genus look highly similar to A. ludens. In its native
range, other Anastrepha species are unlikely to be confused with A. ludens. Outside its native range,
however, several other species (i.e. A. manizaliensis and A. schultzi) are only clearly distinguishable
based on the female terminalia (Norrbom et al., 2005) (Figure 10). Therefore, to guarantee a definite
identification, it is necessary to dissect a female specimen’s aculeus (ovipositor tip) (Figure 11).
www.efsa.europa.eu/publications 17 EFSA Supporting publication 2021:EN-1998Anastrepha ludens survey card Figure 10: Female terminalia of Anastrepha spp. showing eversible membrane and sclerosed hooks: (A) A. ludens, (B) A. obliqua, (C) A. suspensa, (D) A. striata, (E) A. grandis, (F) A. serpentine (Source: Hernández-Ortiz et al., 2020; courtesy of Doctor Vicente Hernández-Ortiz (Research scientist, INECOL) and S y G editores. Reproduced with permission of the authors and publisher) www.efsa.europa.eu/publications 18 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card Figure 11: Aculeus (ovipositor tip) of Anastrepha spp.: (A, B) A. obliqua, (C, D) A. suspensa, (E– G) A. ludens, typical shape, (F) A. ludens, atypical shape, (H) A. serpentine, (I) A. grandis, (J) A. fraterculus, (K) A. fraterculus, (L, M) A. striata (Source: Hernández-Ortiz et al., 2020; courtesy of Doctor Vicente Hernández-Ortiz (Research scientist, INECOL) and S y G editores. Reproduced with permission of the authors and publisher) A protocol for DNA barcoding based on the cytochrome oxidase I ( COI) gene is described in PM 7/129 on DNA barcoding as an identification tool for a number of regulated pests (EPPO, 2016) and can be used for all life stages. This protocol can provide additional information to support morphological identification of A. ludens. The reliability of the COI gene towards species identification has also been tested within the Anastrepha genus. The research shows that, although not applicable for all species, COI may reliably support diagnosis of A. ludens (Barr et al., 2018). Conclusion for pest identification Identification of Anastrepha ludens at the species level requires morphological examination of the aculeus of adult females. A molecular protocol for the identification of A. ludens is also available. www.efsa.europa.eu/publications 19 EFSA Supporting publication 2021:EN-1998
Anastrepha ludens survey card
3. Key elements for survey design
Based on the analyses of the information on the pest and host plants, the different units that are needed
to design the survey have to be defined and tailored to the situation in each Member State. The size of
the defined target population and its structure in terms of number of epidemiological units need to be
known.
When several pests have to be surveyed in the same crop, it is recommended that the same
epidemiological and inspection units are used for each pest in order to optimise the survey programme
as much as possible. This would optimise field inspections as they are organised per crop visit and not
by pest. Table 2 shows an example of these definitions.
Table 2: Example of definitions of the target population, epidemiological unit and inspection unit for
Anastrepha ludens
Definition
Target population All citrus trees in a Member State
A single homogeneous area with citrus trees (e.g. orchard, hectare,
Epidemiological unit
NUTS area)
Inspection unit A single citrus tree or a trap
To design a plant pest survey on Anastrepha ludens, the general guidelines provide further details on
the following steps that will generally be necessary:
1/ Determine the type of survey based on its objectives. For A. ludens, the type of survey will depend
on the pest status (according to ISPM No. 8 (FAO, 2017)) in the area of interest. The objective could
be to substantiate pest freedom, to delimit an outbreak area following an infestation or to determine
the pest prevalence. The next steps deal with the example of substantiating pest freedom.
The overall confidence level and design prevalence of the survey have to be decided by the risk
managers before designing the surveys as they reflect the acceptable level of the risk of infestation of
the host plants by A. ludens. The general guidelines for statistically sound and risk‐based surveys of
plant pests (EFSA, 2020a) provide further details on the choice of these values and the related
consequences in terms of pest surveys.
2/ Define the target population and its size. When determining the target population for surveillance of
A. ludens, the host plants that are relevant for the survey area have to be selected. The size of the
target population should be determined. For example, the target population could be all host plants in
a Member State, but could also be limited to the main hosts (e.g. citrus trees).
3/ Define the epidemiological units. The epidemiological units should be single homogeneous areas that
each contain one at least one individual host plant. These could e.g. be defined as orchards or hectares.
4/ Determine the inspection unit. For example, for a citrus orchard the inspection unit is either an
individual citrus tree or a trap.
5/ Determine the number of inspection units per epidemiological unit. For inspection in a citrus orchard,
this is either the number of host plants or number of traps per epidemiological unit. Given the range of
attraction of food-based attractants in traps for A. ludens, multiple traps may be needed per
epidemiological unit.
6/ Implement the inspections and, if appropriate, the sampling, following the procedures suggested by
the competent authorities, within the epidemiological units and estimate the method effectiveness in
order to determine the overall method sensitivity (sampling effectiveness × diagnostic sensitivity). For
traps, the capture probability directly determines the method sensitivity. For visual examinations, a
representative number of trees should be examined and for suspicious symptoms on fruits these should
be sampled. RiBESS+ can be used to calculate how many inspection units need to be examined or
www.efsa.europa.eu/publications 20 EFSA Supporting publication 2021:EN-1998Anastrepha ludens survey card sampled when using predefined prevalence level (e.g. 1%) to obtain a particular confidence level. This confidence level is in turn needed to calculate the number of sites to be inspected (Step 8). Note that the more units are inspected the higher the confidence will be. The competent authorities need to align the survey efforts with the resources available. 7/ Define the risk factors. A risk factor affects the probability that a pest will be present or detected in a specific portion of the target population. By including the risk factors identified under Section 1.8, the survey focuses mainly on those orchards that are more likely to be infested with A. ludens. It may not always be possible to identify or include a risk factor in the survey design. Risk factors can only be included when both the relative risk and the proportion of the overall plant population to which they apply are known or can be reliably estimated. 8/ Determine the number of epidemiological units to survey. RiBESS+ can be used to determine the number of epidemiological units to survey in order to achieve the objectives of the survey set at Step 1 in terms of confidence level (e.g. 95%) and design prevalence (e.g. 1%), while also including the method sensitivity from Step 6 and the risk factors identified in Step 7. For example, these calculations result in the number of orchards or hectares that need to be surveyed in a Member State in order to state with 95% confidence that the prevalence of A. ludens will be at 1% or below. 9/ Summarise and evaluate the survey design. At this stage, it is necessary to evaluate whether the above steps have resulted in a survey design that matches the available resources, meaning that a feasible number of inspections can be performed or traps can be placed within an acceptable time frame per inspection, and result in a feasible number of samples. If not, available resources should be adjusted. This adjustment would result in a modified survey design using different input parameters of the statistical tool RiBESS+ (e.g. varying the number of components, method sensitivity, etc.). 10/ Integrate the pest-based survey into a crop-based survey (optional). 11/ Allocate the calculated survey effort. The output of RiBESS+ should be allocated proportionally to the host plant population or to the number of epidemiological units in the survey area. In addition, the survey sites should be selected from the list of available locations. 12/ Data collection and survey reporting. Consider which data are needed and how these data will be reported together with the related assumptions. 13/ Plan, develop or update the specific instructions for the inspectors. These activities are not addressed by EFSA and fall within the remit of the competent national authorities. www.efsa.europa.eu/publications 21 EFSA Supporting publication 2021:EN-1998
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