Diagnostic Protocol Anastrepha species 1 Hernández-Ortiz et al.

DIAGNOSTIC PROTOCOL FOR REGULATED PESTS

Fruit of the Genus Anastrepha spp. (: Diptera)

Introduction

The family Tephritidae includes the commonly known “true fruit flies”, which possess a worldwide distribution along temperate, tropical and subtropical regions, comprising around 4,200 species in ca. 500 genera (Norrbom et al. 1999). Because of their food habits in cultivated and wild fruits, some species are considered pests of major economic importance, such as the “Mediterranean fruit ” Ceratitis capitata, native from Africa and currently distributed in the Old World tropics, West Australia, Hawaii, and Central and South America; and the “Oriental fruit fly”, Bactrocera dorsalis, distributed in Asia: East China, Oriental region, and Hawaii (Foote 1984).

Anastrepha Schiner, is the largest and most economically important genus of Tephritidae in the Americas, represented by near 200 species that occur from south of USA (Texas and Florida) to South America in Argentina and the Caribbean Islands (Hernández-Ortiz 1992, Hernández-Ortiz y Aluja 1993, Foote et al. 1993). Nevertheless, only some species are considered pests by the cultivated fruit trees that attack, or by their wide host range, such as the “Mexican fruit fly”, A. ludens (Loew), the “West Indian fruit fly”, A. obliqua (Macquart); the "Caribbean fruit fly", A. suspensa (Loew); the “guava fruit fly”, A. striata Schiner; the "Sapodilla fruit fly", A. serpentina (Wiedemann); the “Melon fruit fly”, A. grandis (Macquart); and the “South American fruit fly”, A. fraterculus (Wiedemann). This genus belongs to the tribe Toxotrypanini, along with other tropical American genera as Toxotrypana Gerstaecker and Hexachaeta Loew, which it have a monophyletic relationship (Foote et al. 1993, Norrbom et al. 2000).

Hosts

Analysis of the relationships between Anastrepha and their host plants are complex, because they have been recorded in about 270 host species from 41 families, but most of this diversity is reported for a few generalist species (Norrbom & Kim 1988). Furthermore, the current information comprise an important quantity of dubious records or infestations produced upon only laboratory conditions. The improvement of host list restricted to natural infestations, denote that we known the hosts for 39.8 % of all known Anastrepha Diagnostic Protocol Anastrepha species 2 Hernández-Ortiz et al. species (Hernández-Ortiz & Aluja 1993).

The major pest species stand out by their polyphagy, because they utilize a wide range of different host plant families, and especially, some exotic species have allowed them expanding their original areas of distribution and their reproductive potential as well, such as Mangifera indica and Citrus spp.. However, they keep on marked preferences for certain native hosts among which could be the original relationships. On this regard, A. suspensa, A. fraterculus and A. striata mainly breed in hosts of the family Myrtaceae, A. ludens in the Rutaceae, A. obliqua in the Anacardiaceae, A. serpentina in the Sapotaceae, and A. grandis in the Cucurbitaceae.

Among native hosts in the American tropics, the family Sapotaceae is recorded with the higher species rate for Anastrepha representing almost all of the species groups, by which have been suggested an ancestral association with this family (Norrbom & Kim 1988). The Sapotaceous fruits are frequent hosts in species of the dentata, leptozona, serpentina, daciformis and cryptostrepha groups. In the same way, the family Myrtaceae is also a very important host group since it have been recorded for about 26 Anastrepha species, particularly in the genera Psidium, Eugenia and Syzygium standing out species of the fraterculus group.

Table 1. Major fruit fly pests of the genus Anastrepha and their main host plants in the Americas Common Anastrepha Synonymy Main Host Plants name species

South Anastrepha Tephritis mellea Walker, 1837 Annona spp (Annonaceae) American fruit fraterculus Trypeta unicolor Loew, 1862 Citrus sinensis (Rutaceae) fly (Wiedemann, Anthomyia frutalis Weyenbergh, 1874 Citrus spp. (Rutaceae) 1830) Anastrepha fraterculus var. soluta Bezzi, Coffea arabica (Rubiaceae) Species 1909 Eriobotrya japonica (Rosaceae) complex Anastrepha peruviana Townsend, 1913 Eugenia spp. (Myrtaceae) Anastrepha braziliensis Greene, 1934 Mangifera indica (Anacardiaceae) Anastrepha costarukmanii Capoor, 1954 Prunus persica (Rosaceae) Anastrepha scholae Capoor, 1955 Psidium guajava (Myrtaceae) Anastrepha pseudofraterculus Capoor, 1955 Psidium spp. (Myrtaceae) Anastrepha lambayecae Korytkowski & Syzygium spp. (Myrtaceae) Ojeda, 1968 Terminalia catappa (Combretaceae)

Melon fruit fly Anastrepha Anastrepha schineri Hendel, 1914 Citrullus lanatus (Cucurbitaceae) grandis Anastrepha latifasciata Hering, 1935 Cucumis sativus (Cucurbitaceae) (Macquart, Cucurbita pepo (Cucurbitaceae) 1846) Cucurbita spp. (Cucurbitaceae) Diagnostic Protocol Anastrepha species 3 Hernández-Ortiz et al.

Mexican fruit Anastrepha Anastrepha lathana Stone Casimiroa edulis (Rutaceae) fly ludens Citrus aurantium (Rutaceae) (Loew, 1873) Citrus paradisi (Rutaceae) Citrus sinensis (Rutaceae) Mangifera indica (Anacardiaceae) Prunus persica (Rosaceae) Sargentia greggii (Rutaceae)

West Indian Anastrepha Anastrepha mombinpraeoptans Sein, 1933 Averrhoa carambola (Oxalidaceae) fruit fly obliqua Anastrepha fraterculus var. ligata Lima, Mangifera indica (Anacardiaceae) (Macquart, 1934 Spondias dulcis (Anacardiaceae) 1835) Anastrepha trinidadensis Greene, 1934 Spondias mombin (Anacardiaceae) Spondias puprurea (Anacardiaceae) Syzygium jambos Myrtaceae)

Sapodilla fruit Anastrepha Urophora vittithorax Macquart, 1851 Chrysophyllum cainito fly serpentina (Sapotaceae) (Wiedemann, Mammea americana (Sapotaceae) 1830) Manilkara zapota (Sapotaceae) Pouteria sapota (Sapotaceae) Pouteria spp. (Sapotaceae)

Guava fruit fly Anastrepha Dictya cancellaria Fabricius, 1805 Psidium guajava (Myrtaceae) striata (see Norrbom et al. 1999) Psidium sartorianum (Myrtaceae) Schiner, 1868 Psidium spp. (Myrtaceae)

Caribbean Anastrepha Anastrepha unipuncta Sein, 1933 Citrofortunella mitis fruit fly suspensa Anastrepha longimacula Greene, 1934 Citrus aurantium (Rutaceae) (Loew, 1862) Citrus sinensis (Rutaceae) Eriobotrya japonica (Rosaceae) Eugenia uniflora (Myrtaceae) Fortunella margarita Manilkara zapota (Sapotaceae) Prunus persica (Rosaceae) Psidium guajava (Myrtaceae) Syzygium jambos (Myrtaceae) Terminalia catappa (Combretaceae)

Biology

Females deposit their eggs inside the fruits, which are hatched by several days, larvae emerged start their process of feeding usually in the pulp of fruit, but also they feed on the seeds in some cases. The number of deposited eggs per fruit is variable, and depends mainly on the own features of the host fruit as size and ripeness (Malavasi et al. 1983, Berrigan et al. 1988). The mature larvae usually come out of the fruit where they feed for Diagnostic Protocol Anastrepha species 4 Hernández-Ortiz et al. pupation in the ground, but the pupation could take place within the feeding fruits in certain cases.

These flies possess life cycles that vary according to the species. Adults usually emerged after a pupal period that it passes between 16 - 25 days approximately. The adults require a period of sexual maturation than usually occurs in 5-20 days after emergency, during this process the flies obtain food from homopteran secretions, bird feces, and juice produced by ripeness fruits (Prokopy & Roitberg 1984).

The adults' longevity is variable, depending on great measure of the ingestion of certain nutrients as amino acids, and of the amount and quality of the food ingested by the larvae. In fruit fly pest species, have been observed that larger size individuals, have a high fertility and expectancy of life than the smaller individuals (Liedo et al. 1991).

Geographic distribution

According to the geographic distribution, Anastrepha presents a Neotropical pattern, typified by native elements from South America that they occur along tropical lowlands, but they are scarcely represented to the north of the continent, especially due to ecological factors. The northern limits of the Neotropical region are located at temperate Mexican areas and the southland of the USA, where the diversity of the genus is lower than in South American countries. Most of the species recorded for the USA occur at the southern region of Texas, while others occur in Florida actually shared with fauna of the Caribbean islands.

The major species diversity of Anastrepha is found in South America with a gradual decreasing toward the north of the continent (Hernández-Ortiz & Aluja 1993). At the North-Center region comprised from south USA to Costa Rica, have been recorded 22.3% of the species, wherein five species groups were absent and three other ones appear scarcely represented; The South American region (from Panama to Argentina) includes 89.3% of the species, where all species groups are represented; while the Caribbean region (including Greater and Lesser West Indies), only 9.1% of the species were recorded. Diagnostic Protocol Anastrepha species 5 Hernández-Ortiz et al.

Table 2. Supraspecific classification of the genus Anastrepha (sensu Norrbom et al, 2000), and recorded species by major geographic regions in the Americas.

Species Groups Known Region USA- Region Region species CRica Panama-South Caribbean America

cryptostrepha 85 5 --

daciformis 13 4 11 4

dentata 9351

benjamini 4--4--

robusta 11 1 10 1

schausi 41 3 --

punctata 4--4--

leptozona 52 5 --

mucronota 31 4 27 5

grandis 8--8--

doryphoros 2--2--

spatulata 10 6 9 --

ramosa 2--2--

pseudoparallela 20 2 20 1

serpentina 4231

striata 3131

fraterculus 29 11 24 4

I. sedis 32 4 31 --

Totals 199 46 176 18

Identity

Name: Anastrepha Schiner, 1868 Synonyms: Acrotoxa Loew, 1873 Pseudodacus Hendel, 1914 Phobema Aldrich, 1925 Lucumaphila Stone, 1939 Taxonomic position: Insecta: Diptera: Tephritidae, Diagnostic Protocol Anastrepha species 6 Hernández-Ortiz et al.

Detection

The fruit flies can be detected either inside the fruits as larvae, or as pupae in the boxes or containers where they are transported, but also by means of trapping systems in susceptible areas of introduction of the pest.

Inspection of fruits. Often infested materials can be found in imported or exported shipments of fruits, in baggage of passengers, or else in airplanes or terrestrial transport. Must be inspected fruits with soft areas, darkened stains, rottings, orifices or injuries that may be originated by the oviposition of females or by the larvae nutrition. If you observed larvae's holes of exit, you must inspect the fruit containers because it is suspected to find pupae.

Inspection of traps. The monitoring systems established for fruit flies detection, either at growing fruit regions, or at border areas between countries, you require of the utilization of McPhail traps baited with food attractants for adults' capture. The utilized baits must be a rich sources in ammonium, recognized and approved internationally. The specific methods of baited, location, and time of service of the traps must be in agreement with fitosanitary regulations in use for each country.

Handling the biological sample. Even though the third instar larvae of some fruit flies can be identified with a reasonable degree of certainty, also it is certain that doubts related to determination of some other species can exist, for which it is convenient to enable their full development to adult stage. Full larvae development will only be able to take place at laboratories approved by the fitosanitary procedures of the involved countries. In case of finding larvae, it is recommended to preserve some specimens in alcohol 70%, while the remaining material, so as mature larvae or pupae found it must be placed in containers made suitable with a sterile substratum, and lidded supplied of fine mesh, that allow obtain adult specimens for their identification.

Rearing Adults

Materials

Cages with wire netting, plastic, or other appropriate materials Sterile substratum for pupation (e.g., vermiculite, sand or sawdust) - Place the fruits on the cages whose bottom must be covered for some substratum that it enable normal development of pupae. - Once larvae come out of the fruit, they moved to the substratum for pupation. It is commendable that each originating different sampled fruits should be handled of Diagnostic Protocol Anastrepha species 7 Hernández-Ortiz et al.

individual way. - Each sample must come under review every day and gathering pupae, separating them in containers with the same substratum, and lidded tight-lipped but that it enable aeration. - Once the adults emerged, they must remain alive for 48-72 hours to assure that the tegument and wings acquire the rigidity and characteristic coloration of the species. - Kill the adults in lethal camera with ethyl acetate and preserving in alcohol 75%.

Techniques of Preservation and Analysis

Materials

Stereomicroscope (45X magnification at least) Compound microscope of high resolution (400X at least), provided with ocular micrometer for the measurement of structures Hotplate Fine dissection forceps Fine surgery scissors pins (sizes: 00, 0 and 1) Precipitated glasses (25 ml) Slide and cover slides Hermetic vials (15-25 ml) Data labels Microvials for genitalia 10% KOH solution Ethyl alcohol (50%, and 70%) Canada Balsam, or Euparal Xylene Glycerine

Procedure for preparation of larvae

- The alive larvae settle down into hot water for 2-4 minutes, afterwards let themselves to get cold. - Immerse in alcohol 50% for 15 - 30 minutes, and finally transferring into hermetic vial with alcohol 70%, labeling with all data sample. - For mounting slides, make two transverse incisions in the larva, first one behind of the cephalic region and the anterior spiracles; and the later before the caudal segment. - Put the larva into a test tube containing 10%NaOH solution and heat in boiling Diagnostic Protocol Anastrepha species 8 Hernández-Ortiz et al.

water bath for 10-15 minutes. - Place the specimen in distilled water under the stereomicroscope, and clean all the internal contents even to be allow observe the transparent cuticle, the oral opening, the cephalopharyngeal skeleton and the anterior spiracles in the former incision, as well as the posterior spiracular plate and anal lobes in the latter incision. - Permanent slides can be made using Canada Balsam or Euparal. - Cleaned structures must be transferred to gradual alcohols for dehydration as follows: 25 minutes in each alcohol 50%, 75%, 100%. - Structures must be placed in xylene by only 3-5 minutes and immediately transferred to slide with 1-2 drops of Canada Balsam. Slides must be dried by several days. - For Euparal mounting slides, structures can be transferred from alcohol 100% directly to Euparal. - Label the mounting slide and observing at microscope.

Procedure for preparation of adults

- For the species recognition in adult stage, the preservation of the wings and the aculeus or ovipositor is needed. - Examination of the aculeus must be held at 400X magnification. It is commendable that both structures (wing and acuelus) of each specimen should be mounted separately, but in one single slide.

Aculeus or Ovipositor: - In specimens preserved in alcohol 70%, take a female cutting off all of the abdomen, so that enclose the oviscape (syntergosternite 7), the eversible membrane and the aculeus. - In preserved dry specimens (pinned specimens), the abdomen must be cut off with fine dissection scissors. - Place the abdomen 10%NaOH solution and heat in boiling water bath for 10-15 minutes. - Wash the structure into distilled water, removing the internal contents under the stereomicroscope helped with dissection forceps. - Expose the aculeus and the eversible membrane. - For dry specimens, at this step it is possible analyze the aculeus directly with 1-2 drops of glycerine covering and observe under microscope. Further this structure can be transferred to microvial with glycerine and pinned under the mounted dry specimen. - For permanent slides dehydrate with gradual alcohols (50%, 75%, 100%) for 25 minutes in each case. - Place the aculeus in xylene by only 3-5 minutes and immediately transferred to slide Diagnostic Protocol Anastrepha species 9 Hernández-Ortiz et al.

with 1-2 drops of Canada Balsam. Slides must be dried by several days. - For Euparal mounting slides, the aculeus can be transferred from alcohol 100% directly to Euparal. - Label the mounting slide and observing at microscope.

Wings: - Cut off the right wing (preferably) from its base. - Dehydrate with gradual alcohols (50%, 75%, 100%) for 25 minutes in each case. - Place the structure with some xylene drops for 3-5 minutes. - Mount in slides with 2 drops de Canada Balsam, placing the wing in dorsal view. - Label the slide and observing at microscope.

Identification

Key to third instar larvae for Anastrepha species of economic significance (Adapted from Steck et al. 1990)

1 Posterior spiracles prominently raised from the body surface; or most body segments with conspicuous setae or processes; or posterior spiracular openings sinuous...... Not Tephritidae Posterior spiracles nearly flush with body surface; tubercles, if present, on caudal segment only; posterior spiracular slits elongate or oval (fig. 6)...... (Tephritidae) 2

2 With prominent chitinized teeth (stomal guards) adjacent to oral opening, and strongly developed tubercles on caudal segment; or dental sclerite conspicuous; or larva taken from papaya with caudal tubercles lacking and caudal sensilla all very reduced...... Other Tephritidae (not Anastrepha) Lacking stomal guards; caudal tubercles at most moderately developed; dental sclerite lacking or inconspicuous...... (Anastrepha) 3

3 Dorsal spinules present at least on two or more abdominal segments, separate, conical, in fewer than 5-6 rows on thoracic segments T2 and T3; posterior spiracular processes SP-1 and SP-IV with average of 6 or more trunks with bristle length 1/3 or more times length of spiracular opening...... 4 Dorsal spinules absent in all abdominal segments, but if present, only in segment A1 (some specimens of A. ludens)...... 5

4 Anterior spiracle with 28-37 tubules (fig. 23); larvae breed in fruits of Cucurbitaceae (Panama to Argentina)...... Anastrepha grandis Diagnostic Protocol Anastrepha species 10 Hernández-Ortiz et al.

Anterior spiracle with 12-23 tubules (fig. 21); larvae mainly breed in fruits of Myrtaceae (particularly in Psidium spp.)...... Anastrepha striata

5 Dorsal spinules present on thoracic segment T3; ...... 6 Dorsal spinules absent on thoracic segment T3 ...... 7

6 Dorsal spinules present in abdominal segment A1; oral ridges 11-17 rows with margins entire; anterior spiracle with 12-20 tubules (fig. 13); posterior spiracular slits 3.1-4.6 X longer than wide (fig. 14). Larvae frequently breeding in fruits of Citrus spp. (Rutaceae) or Mangifera indica (Southern Texas to Costa Rica)...... Anastrepha ludens Dorsal spinules absent in all abdominal segments; oral rigdes 8-11 rows with stout, bluntly rounded, widely spaced teeth; anterior spiracles 9-15 tubules (fig. 17); posterior spiracular slits 2.5-3.5 X longer than wide (fig. 18) (Caribbean region) ...... Anastrepha suspensa

7 Posterior spiracular processes SP-I and SP-IV with 6-9 short trunks (fig. 20); oral ridges usually 12-14 rows; anterior spiracle with 13-19 tubules in a single row (fig. 19); anal lobes usually bilobed. Larvae mainly breeding in fruits of Sapotaceae ...... Anastrepha serpentina Posterior spiracular processes SP-I and SP-IV with 10-17 long trunks; oral ridges 7- 10 rows; anterior spiracle with 9-18 tubules in a single row; anal lobes usually entire. Larvae fraquently breeding in fruits of Myrtaceae and Anacardiaceae, rarely on Sapotaceae...... 8

8 Posterior spiracular processes SP-II usually with 3-6 trunks (fig. 16); posterior spiracular slits 3-4.5 X longer than wide (fig. 15) ...... Anastrepha obliqua Posterior spiracular processes SP-II usually with 6-9 trunks; posterior spiracular slits 2.5-3.5 X longer than wide (fig. 6)...... Anastrepha fraterculus (species complex) Diagnostic Protocol Anastrepha species 11 Hernández-Ortiz et al.

Identification of Adults

Head usually yellow with 3-5 frontals and 2 orbital setae, sometimes posterior orbital seta absent; ocellar seta very weak or indistinct; postocellar, medial and lateral vertical setae present. Macrosetae of thorax usually black sometimes red brown and rarely golden yellow; scutum yellow, sometimes with dark brown or black stripes or spots; mesonotum with following setae present: 1 postpronotal, 2 notopleurals, 1 presutural supra-alar, 1 postsutural supralar, 1 postalar, 1 intra-alar, 1 dorsocentral, 1 acrostichal, and 2 scutellars.

Wings with subcostal break present; crossvein R-M placed after middle of discal cell length (dm); basal cubital cell (bcu) with a posteroapical extension moderately developed; vein M conspicuosly curved forward apically. Wing pattern yellow or sometimes brown colored banded forming typical pattern as follows: C-band on basal costal margin including base of vein R1, subcostal cell and the pterostigma; S-band extended from apex of cell bcu across cell dm, crossvein R-M reaching costal margin, and continued to apex of wing; and V-band forming an inverted V shaped, composed by the proximal arm (subapical band) along vein DM-Cu, and the distal arm (posterior apical band) arising from cell m, both are convegent in cell R.4+5 The typical wing pattern could be modified in some economic important species (see key to species).

Male terminalia: epandrium broad in lateral view with lateral surstylus short or elongated; medial surstylus near as long as lateral surstylus with two prensisetae developed; proctiger membranous sometimes sclerotized below; aedeagus elongated usually near as long as aculeus length of female; glans weakly sclerotized with an internal sclerite T-shaped, sometimes absent in not economically important species.

Female terminalia: length of syntergosternite 7 variable, inside with an eversible membrane with dorsal hook-like sclerotized plates (also named rasper); aculeus or ovipositor connected to eversible membrane, well sclerotized with the aculeus tip sometimes serrated on lateral margins.

Key to adult fruit fly genera of economic significance or commonly found in the Neotropics

1 The following characters present: All postocular setae unicolorous, slender and pointed apically; dorsocentral seta absent or extremely reduced, but if present and well developed, then it is located well behind to level of postsutural supra-alar seta (usually closer to level of postalar seta); wing pattern usually banded or diffuse, never reticulated...... 2 Diagnostic Protocol Anastrepha species 12 Hernández-Ortiz et al.

At least one of following characters present: postocular setae bicolored, mixed black with white flattened setae, but if not, then dorsocentral setae located before to level of postsutural supra-alar seta (usually closer to transverse suture), or sometimes a second pair of presutural dorsocentral seta is present; wing pattern variable. . . . 9

2 Macrosetae of head and thorax greatly reduced or absent, at least following setae absent: frontals, postpronotals, presuturals, supra-alars and dorsocentrals; wing pattern diffuse usually with a yellow or brownish stripe along anterior marging of wing, and on cell bcu, and/or on crossvein dm-cu...... 3 Macrosetae of head and thorax well developed, at least the following setae present: frontals, postpronotals, presuturals, supra-alars and dorsocentrals; wing pattern usually banded or hyaline spotted...... 5

3 Abdominal syntergite 1+2 elongate and petiolate (basal half more narrowed than

rest of abdomen); vein R2+3 with 3 sharp bends, often with spurs veins; scutum with weak medial longitudinal depression; cell bcu with inferior apical corner few more than one half long that body cell; oviscape of female at least as long as rest of abdomen, often strongly curved ventrally...... Toxotrypana Gerstaecker

Abdominal syntergite 1+2 short, not petiolate; vein R2+3 without sharp bends or spur veins; scutum without medial depression; inferior apical corner of cell bcu usually very narrow and elongated (close two times longer than body cell length); oviscape of female variable, usually shorter than rest of abdomen, not curved ventrally . 4

4 Abdomen with all tergites separate, view from side to see overlapping sclerites...... Bactrocera Macquart Abdomen with all tergites fused into a single plate, at most with smooth transverse lines marking the boundaries of each segment, view from side no sclerites overlap the next...... Dacus Fabricius

5 Two pairs of scutellar setae...... 6 Three pairs of scutellar setae...... 8

6 Vein M curved forward apically, meeting costa in gradual curve (except in some not economically important species); ocellar seta very weak sometimes indistinct;

typical wing pattern with costal band extended to apex of vein R1; S-band extending

obliquely from cell bcu, across vein R-M, to wing margin of cell r1, then bordering costa to wing apex; the subapical and posterior apical bands forming inverted V- band (distal arm sometimes absent)...... Anastrepha Schiner Vein M not curved forward apically, meeting costa (perpendicular) at distinct angle; ocellar seta aften strong and well developed; wing pattern different from above. 7 Diagnostic Protocol Anastrepha species 13 Hernández-Ortiz et al.

7 Wing with small dark spots in cells bc, c, br, bm, and bcu; crossvein R-M near midlength of cell dm; wing pattern with yellow or brown band from the pterostigma

to near wing margin on cell cu1 crossing R-M; band covering crossvein dm-cu; inferior apical corner of cell bcu narrower near base than near middle; scutellum bulky and shining usually marked with yellow and black; male with anterior orbital seta proclinate and expanded apically; third apical segment of antenna rounded apically...... Ceratitis MacLeay Wing not dark spotted on basal cells; wing pattern usually with three dark transverse bands, frequently subcostal band present; scutellum flattened dorsally, opace; male with anterior and posterior orbital setae usually reclinate pointed apically; third apical segment of antenna usually dorsoapically pointed...... Rhagoletis Loew

8 Anepisternum with a strong seta anterior to phragma; veins BCu and Cu1 always setulose dorsally; usually two postpronotal setae; wing pattern usually reticulated with hyaline spots on surface...... Blepharoneura Loew Anepisternum without a strong seta anterior to phragma; vein BCu sometimes

setulose dorsally; vein Cu1 always bare; one postpronotal seta; wing pattern usually banded, sometimes broadly dark on basal half of wing ...... Hexachaeta Loew

9 Wing pattern usually reticulated, hyaline spotted, or stellate marking on apical half, postocular setae mixed (black and white), sometimes including the orbital setae ...... Other genera Tephritinae Wing pattern usually banded, postocular setae unicolorous (yellow or black) apically pointed and not flattened...... 10

10 Wing pattern with 3-4 yellow oblique bands on same direction; scutum yellow with paired posterolateral black points; abdominal tergites yellow, usually with a line of lateral black points...... Tomoplagia Cresson Wing pattern with dark brown bands, at least one of them not obliquely directed; scutum and abdominal tergites mainly dark, or without dark points...... Other genera Tephritinae Diagnostic Protocol Anastrepha species 14 Hernández-Ortiz et al.

Key to adult Anastrepha species of economic significance or commonly found in the neotropics

1 Costal band of wing complete along costa from wing base to apex, sometimes diffuse; posterior orbital seta usually absent...... 2

Costal band of wing interrupted at end of vein R1 by a hyaline mark well delimited

on cell r1; posterior orbital seta present...... 7

2 Distal arm of V-band absent (sometimes the proximal arm also absent); scutum with black or brown reddish stripes or spots forming patterns...... 3 Distal arm of V-band present...... Other species

3 Basal half of S-band complete from apex of cell bcu through discal cell and crossvein R-M, connecting anteriorly with C-band; aculeus of female extremely long (usually > 0.10 mm wide); glans of female present...... 4 Basal half of S-band absent or interrupted along discal cell; aculeus of female long but extremely narrow (usually < 0.05 mm wide); glans of male absent...... 6

4 Cell r2+3 with a hyaline area close to crossvein R-M; vein R2+3 strongly sinuous (fig. 53); eversible membrane of female with dorsal sclerotized short scales broadly expanded along membrane; aculeus length 5.6 - 9.2 mm long (fig. 38)...... Anastrepha bezzii Lima

Cell r2+3 dark (pigmented) basally to level of crossvein R-M; vein R2+3 almost straight in all length...... 5

5 All following characters present: Basal half of S-band (on discal cell) continuous

from apex of cell cup through crossvein R-M to connecting with C-band; cell r2+3 completely darkened (pigmented) in all length; cell br broadly hyaline between veins BM-Cu and R-M (fig. 54); abdominal tergites yellow, scutum with dark brown dorsocentral stripes; aculeus 5.3 - 6.2 mm long, with aculeus tip margins not serrated (fig. 39-40) ...... Anastrepha grandis (Macquart)

At least one of the following characters present: Basal half of S-band absent; cell r2+3 with a broad subapical hyaline area; cell br completely dark; abdominal tergites dark except by a medial yellow mark, or scutum without dark dorsocentral stripes; aculeus variable...... Other species

6 C-band present on cell r1+2, while cell r2+3 hyaline; proximal arm of V-band present on vein DM-Cu...... Anastrepha daciformis Bezzi Diagnostic Protocol Anastrepha species 15 Hernández-Ortiz et al.

At least one of following characters present: C-band occupying all of cell r2+3; abdominal tergites with dark brown stripes on middle; proximal arm of V-band diffuse...... Other species

7 Abdominal tergites with dark brown or black stripes and/or scutum with dorsocentral stripes forming a well differentiated pattern...... 8 Abdominal tergites yellow unicolorous; scutum yellow or orange, dark stripes absent except sometimes along scuto-scutellar suture...... 10

8 Wing pattern dark brown or black; distal arm of V-band absent (fig. 47); abdominal tergites with dark brown stripes; broad black spots on pleuron contrasting with yellow markings; aculeus 2.8 - 3.7 mm long, with aculeus tip finely serrated (fig. 32)...... Anastrepha serpentina (Wiedemann) Wing pattern mostly yellow; distal arm of V-band present or weakly present basally; abdominal tergites yellow; scutum with two broad “U” shaped dorsocentral stripes connected on posterior margin...... 9

9 Dorsocentral stripes of scutum without setulae in a small area along transverse suture, but with dense white microsetae contrasting with black microsetae on this area; aculeus wide, 2.05 - 2.30 mm long (fig. 37)...... Anastrepha striata Schiner Dorsocentral stripes of scutum with setulae on the transverse suture, with only few white microsetae on this area...... Anastrepha bistrigata Bezzi

10 Subscutellum and mediotergite, or just one of them with lateral black markings; and/or aculeus tip at most with 14 marginal teeth on each side (A. suspensa) ...... 11 Subscutellum and mediotergite completely yellow (with no dark lateral markings); aculeus tip usually with lateral margins with numerous fine serrations (always more than 20 teeth on each side)...... 16

11 Aculeus length more than 2.0 mm; aculeus tip elongated, lateral margins with few

short teeth; S-band and V-band always separated on cell r2+3 ...... 12 ' Aculeus length less than 2.0 mm (usually between 1.4 - 1.9 mm); aculeus tip short and wide, with big teeth on sides...... 13

12 Aculeus 3.1 - 4.9 mm long; acuelus tip length 0.34-0.38 mm, with a moderate constriction near midlength; marginal teeth more or less symmetric and apically rounded (fig. 41); black markings of subscutellum evident and sometimes extended into mediotergite narrow gradually...... Anastrepha ludens (Loew) Diagnostic Protocol Anastrepha species 16 Hernández-Ortiz et al.

Aculeus 2.1 - 2.9 mm long (sometimes reaching 3.4 mm in specimens from Los Andes); aculeus tip length about 0.40 mm, without any constriction; lateral margins with few small irregular teeth (fig. 42); black markings of subscutellum small and extended into mediotergite of similar wide than precedent...... Anastrepha distincta Greene

13 Anterior apical band (Distal arm of S-band) very broad, usually reaching the apex of vein M; aculeus 1.4 - 1.6 m long; V-band broad and complete, always connected to S-band (fig. 59); usually a big rounded spot present on middle of scuto-scutellar suture; mediotergite yellow or black spotted on sides...... Anastrepha suspensa (Loew) Anterior apical band (distal arm of S-band) normally developed, never reaching the apex of vein M; aculeus length variable; V-band usually connected anteriorly to S- band, if not, both arms of V-band are separated; black spot on middle of scuto- scutellar suture absent but if present then not rounded; mediotergite always with black lateral spots...... 14

14 Subscutellum yellow, only the mediotergite with black lateral markings on sides (fig. 28); black spot on scuto-scutellar suture absent; aculeus tip 0.16 - 0.20 mm long, with lateral serrations on 2/3 or 4/5 of apex (fig. 44)...... Anastrepha obliqua (Macquart) Subscutellum and mediotergite (both) with black lateral markings on sides (fig. 29); black spot on scuto-scutellar suture usually present (sometimes absent in the A. fraterculus complex); aculeus tip length variable...... 15

15 All of following characters present: aculeus 1.4 -1.9 mm long; aculeus tip 0.20 - 0.28 mm long; lateral margins with 8 - 14 teeth occupying 2/5 to 3/5 of aculeus tip length (fig. 43)...... Anastrepha fraterculus (Wiedemann) (Species complex) Aculeus 1.43 - 1.56 mm long); aculeus tip 0.17 - 0.20 mm long; lateral margins with 6 - 9 teeth occupying 2/3 or more of aculeus tip length...... Anastrepha sororcula Zucchi

16 Scuto-scutellar suture with a nearly circular medial black spot; cells r1 and r2+3 usually with small marginal hyaline areas (fig. 51); aculeus tip lanceolate with numerous small serrations (fig. 36)...... Anastrepha spatulata Stone Medial black spot of scuto-scutellar suture absent, but if present then its diffuse of

irregular form; cells r1 and r2+3 completely pigmented on apical margins...... 17 Diagnostic Protocol Anastrepha species 17 Hernández-Ortiz et al.

17 Aculeus tip long, not expanded laterally at base, at least two times longer than wide, tapering gradually to apex; marginal teeth small and numerous but not reaching the base of aculeus tip...... 18 Aculeus tip expanded laterally, almost triangular shaped at beginning of serrated portion, sides of tip sometimes curved dorsally...... Other species

18 Vein M strongly curved forward reaching the anterior apical band; wing pattern with narrow bands with C-band, S-band and V-band always separated among them; distal arm of V-band weak and separated anteriorly from proximal arm, sometimes absent (fig. 46)...... Anastrepha leptozona Hendel Vein M moderately curved anteriorly, not reaching the anterior apical band; wing pattern with broad bands, with C-band, S-band and V- band very close among them, sometimes weakly connected among them; distal arm of V-band broad and complete, always connected anteriorly to proximal arm (fig. 50)...... Anastrepha chiclayae Greene

Records:

The following records are to be kept: - Scientific name of the pest identified. - code or reference number of the sample (for traceability). - nature of the infected/infested material including scientific name of host where applicable. - origin of the infected/infested material. - description of signs or symptoms (including photographs where relevant) - methods, including controls, used in the diagnosis and the results obtained with each method. - for morphological methods, measurements , drawings or photographs of the diagnostic features (where relvant), if applicable the developmental stage. - for biochemical and molecular methods, documentation of the test results such as photographs of the diagnostic gels, ELISA, printouts of results, on which the diagnosis was based. - where appropriate, the magnitude of any infection/infestation (how many individuals pests found; how much damaged tissue). - the name of the laboratory and, where appropriate, the name of the person(s) responsible for and/or who performed the diagnosis. The retention of culture(s) of the pest, preserved/mounted specimens, or test materials (e.g., photographs of gels, ELISA plate printout results) is recommended in cases of non- Diagnostic Protocol Anastrepha species 18 Hernández-Ortiz et al. compliance (ISPM No. 13: Guidelines for the notification of non-compliance and emergency action) and where pests are found for the first time.

Further information:

Instituto de Ecología A.C., Departamento de Entomología. Apartado Postal 63. Xalapa, Veracruz, CP 91000, Mexico. [email protected]. of adults. Systematic Entomology Lab./ Entomology. Smithsonian Institution. Washington DC USA. E-mail: [email protected]. Taxonomy of adults. Escuela Superior de Agricultura Luiz de Queiroz (ESALQ). Piracicaba, Brazil. E-mail: [email protected]. Taxonomy of adults. Instituto de Entomología, Universidad Metropolitana de Ciencias de la Educación. Santiago, Chile. E-mail: [email protected]. Taxonomy of larvae.

Acknowledgements

This protocol was drafted by: Vicente Hernández-Ortiz, Instituto de Ecología A.C. Departamento de Entomología. Apartado Postal 63. Km 2.5 carretera antigua a Coatepec # 351, Congregación El Haya. CP. 91070 Xalapa, Veracruz, Mexico. [email protected] Norma Vaccaro, Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Concordia. C.C. No. 34, Concordia, Entre Ríos, Argentina. Alicia Basso, Universidad de Buenos Aires, Facultad de Agronomía. Av. San Martín # 4453, Buenos Aires, Argentina.

References

Berg, G.H. 1979. Clave ilustrada de larvas de moscas de la fruta de la familia Tephritidae. Organismo Internacional Regional de Sanidad Agropecuaria, El Salvador, C. A., 36 pp. Carroll, L.E., A.L. Norrbom, M.J. Dallwitz, & F.C. Thompson. 2004. Pest fruit flies of the world – larvae. Version: 13th April 2005. http://delta-intkey.com. Carroll, L.E. & R.A. Wharton. 1989. Morphology of the immature stages of Anastrepha ludens (Diptera: Tephritidae). Annals of the Entomological Society of America 82: 201-214. Foote, R.H. 1984. Family Tephritidae (Trypetidae), pp: 66-148. In: Catalogue of Palearctic Diptera Vol. 9 Micropezidae-Agromyzidae, A. Soos & L. Papp (Eds.). Elsevier, Amsterdam. Diagnostic Protocol Anastrepha species 19 Hernández-Ortiz et al.

Foote, R.H., F.L. Blanc & A.L. Norrbom, 1993. Handbook of the fruit flies (Diptera: Tephritidae) of America North of Mexico. Comstock Publ., Cornell University Press, 571 pp. Frías, D., V. Hernández-Ortiz, N. Baccaro, A. Bartolucci & L. Salles. 2006. Comparative morphology of immature stages in some frugivorous species of fruit flies (Diptera: Tephritidae). In: Biotaxonomy of the Tephritoidea, A. Freidberg (Ed.) (In press). Hernández-Ortiz, V. 1992. El género Anastrepha Schiner en México. Taxonomía, distribución y sus plantas huéspedes. Instituto de Ecología, Publicación # 33, Xalapa, México. 167 pp. Hernández-Ortiz, V. & M. Aluja. 1993. Listado de especies del género neotropical Anastrepha (Diptera: Tephritidae) con notas sobre su distribución y plantas hospederas. Folia Entomológica Mexicana 88: 89-105. Hernández-Ortiz, V., J.A. Gómez-Anaya, A. Sánchez, B.A. McPheron & M. Aluja. 2004. Morphometric analysis of Mexican and South American populations of the Anastrepha fraterculus complex (Diptera: Tephritidae) and recognition of a distinct Mexican morphotype. Bulletin of Entomological Research 94: 487-499. Malavasi, A., J.S. Morgante & R.J. Prokopy. 1983. Distribution and activities of Anastrepha fraterculus (Diptera:Tephritidae) flies on host and nonhost trees. Annals of the Entomological Society of America 76: 286-292. Norrbom, A.L., 1991. The species of Anastrepha (Diptera: Tephritidae) with a grandis-type wing pattern. Proceedings of the Entomological Society of Washington 93: 101-124. Norrbom, A.L., L.E. Carroll & A. Freidberg. 1998. Status of Knowledge, pp: 9-47. In: Fruit fly expert identification system and systematic information database, F.C. Thompson (Ed.). Backhuys Publishers, Leiden, Netherlands. Norrbom, A.L. & K.C. Kim. 1988. A list of the reported host plants of the species of Anastrepha (Diptera: Tephritidae). United States Department of Agriculture, APHIS 81-52, 114 pp. Norrbom, A.L., R.A. Zucchi & V. Hernández-Ortiz, 2000. Phylogeny of the genera Anastrepha and Toxotrypana (Trypetinae: Toxotrypanini) based on morphology, pp: 299-342. In: Fruit flies (Tephritidae): phylogeny and evolution of behavior, M. Aluja & A.L. Norrbom (Eds.). CRC Press, Boca Raton, USA. Prokopy, R.J. & B.D. Roitberg. 1984. Foraging behavior of true fruit flies. American Scientist 72: 41-49. Steck, G.J., L.E. Carroll, H. Celedonio-Hurtado & J. Guillén-Aguilar. 1990. Methods for identification of Anastrepha larvae (Diptera: Tephritidae), and key to 13 species. Proceedings of the Entomological Society of Washington 92: 333-346. Steck, G.J. & R.A. Wharton. 1988. Description of immature stages of Anastrepha interrupta, A. limae, and A. grandis (Diptera: Tephritidae). Annals of the Entomological Society of America 81: 994-1003. Stone, A. 1942. The fruit flies of the genus Anastrepha. United States Department of Agriculture Miscellaneous Publication 439: 1-112. Diagnostic Protocol Anastrepha species 20 Hernández-Ortiz et al.

White, I.M. & M.M. Elson-Harris, 1992. Fruit flies of economic significance: their identification and bionomics. CAB International, Wallingford , United Kindom, 601 pp. Zucchi, R.A. 2000. Taxonomia, pp: 13-24. In: Moscas-das-frutas de importância econômica no Brasil. Conhecimento básico e aplicado, A. Malavasi & R.A. Zucchi (Eds.). Holos Editora, Riberao Preto, Brasil. Diagnostic Protocol Anastrepha species 21 Hernández-Ortiz et al.

Figures 1 - 6. Morphology of third instar larvae (A. fraterculus from Mexico): 1) head, lateral view: an= antenna; mh= mouthooks; mso= maxillary sensory organ; or= oral ridges; sso= stomal sensory organ. 2) anteno-maxillary complex; 3) anterior spiracle; 4) head, ventral view; 5) anal lobes, lateral view; 6) posterior spiracular plates: sl= spiracular slits; SP-I to SP-IV= spiracular processes. Diagnostic Protocol Anastrepha species 22 Hernández-Ortiz et al.

Figures 7 - 12. Anastrepha species, cephalopharyngeal skeleton of third instar larvae: 7) A. ludens; 8) A. obliqua; 9) A. suspensa; 10) A. serpentina; 11) A. striata; 12) A. grandis. (Redrawn from Carroll et al. 2004). Diagnostic Protocol Anastrepha species 23 Hernández-Ortiz et al.

Figures 13 - 24. Anastrepha species, anterior and posterior spiracles of third instar larvae: 13-14) A. ludens; 15-16) A. obliqua; 17-18) A. suspensa; 19-20) A. serpentina; 21-22) A. striata; 23-24) A. grandis. (Redrawn from Carroll et al. 2004). Diagnostic Protocol Anastrepha species 24 Hernández-Ortiz et al.

Figures 25 - 30. Adult morphology of Anastrepha species: 25) head, lateral view; 26) thorax, dorsal view; 27-29) scutellum and mediotergite, posterior view; 30) wing, dorsal view. Abbreviations head: frs= frontal setae; iv= inner vertical; ors= orbital seta; ov= outer vertical; poc= postocular setae; poce= postocellar seta. Abbreviations quetotaxy of thorax: ac= acrostichal; asa= anterior supra-alar; dc= dorsocentral; in= intra-alar; ntp= notopleural; pa= postalar; ppn= postpronotal; psa= posterior supra-alar; sc= scutellar. Abbreviations wing: Cb= Costal band; Sb= S-band; Vb= V-band. (Redrawn from Hernández-Ortiz, 1992). Diagnostic Protocol Anastrepha species 25 Hernández-Ortiz et al.

Figures 31 - 37. Shape of the aculeus tip in Anastrepha species: 31) A. leptozona; 32) A. serpentina; 33) A. bicolor; 34) A. pallens; 35) A. chiclayae; 36) A. spatulata; 37) A. striata. (Redrawn from Hernández- Ortiz, 1992). Diagnostic Protocol Anastrepha species 26 Hernández-Ortiz et al.

Figures 38 - 45. Shape of the aculeus tip in Anastrepha species: 38) A. bezzii; 39-40) A. grandis, ventral and lateral view; 41) A. ludens; 42) A. distincta; 43) A. fraterculus; 44) A. obliqua; 45) A. suspensa. (Redrawn from Hernández-Ortiz, 1992, except figs. 29-30 from Norrbom, 1991; and fig. 35 from White & Elson-Harris, 1992). Diagnostic Protocol Anastrepha species 27 Hernández-Ortiz et al.

Figures 46 - 59. Wing pattern in Anastrepha species: 46) A. leptozona; 47) A. serpentina; 48) A. bicolor; 49) A. pallens; 50) A. chiclayae; 51) A. spatulata; 52) A. striata; 53) A. bezzii; 54) A. grandis; 55) A. ludens; 56) A. fraterculus, Mexico; 57) A. fraterculus, Brazil; 58) A. obliqua; 59) A. suspensa.