Development and evaluation of pupal color‐based genetic sexing strains in Anastrepha obliqua (Diptera: Tephritidae) - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Insect Sci . 2025 Sep 8;33(2):678–690. doi: 10.1111/1744-7917.70163 Search in PMC Search in PubMed View in NLM Catalog Add to search Development and evaluation of pupal color‐based genetic sexing strains in Anastrepha obliqua (Diptera: Tephritidae) Daisy P Cárdenas‐Enríquez Daisy P Cárdenas‐Enríquez 1 Programa Operativo Moscas, SADER/SENASICA‐IICA, Metapa de Domínguez, Chiapas, Mexico Find articles by Daisy P Cárdenas‐Enríquez 1 , Víctor García‐Martínez Víctor García‐Martínez 1 Programa Operativo Moscas, SADER/SENASICA‐IICA, Metapa de Domínguez, Chiapas, Mexico Find articles by Víctor García‐Martínez 1 , Jorge Ibáñez‐Palacios Jorge Ibáñez‐Palacios 1 Programa Operativo Moscas, SADER/SENASICA‐IICA, Metapa de Domínguez, Chiapas, Mexico Find articles by Jorge Ibáñez‐Palacios 1 , Brenda Torres‐Huerta Brenda Torres‐Huerta 1 Programa Operativo Moscas, SADER/SENASICA‐IICA, Metapa de Domínguez, Chiapas, Mexico Find articles by Brenda Torres‐Huerta 1 , Maria F Ruiz‐Pérez Maria F Ruiz‐Pérez 1 Programa Operativo Moscas, SADER/SENASICA‐IICA, Metapa de Domínguez, Chiapas, Mexico Find articles by Maria F Ruiz‐Pérez 1 , José S Meza José S Meza 1 Programa Operativo Moscas, SADER/SENASICA‐IICA, Metapa de Domínguez, Chiapas, Mexico Find articles by José S Meza 1, ✉ Author information Article notes Copyright and License information 1 Programa Operativo Moscas, SADER/SENASICA‐IICA, Metapa de Domínguez, Chiapas, Mexico * Correspondence: José S. Meza, Programa Operativo Moscas, SADER/SENASICA‐IICA, Camino a los Cacaotales S/N, CP 30860 Metapa de Domínguez, Chiapas, Mexico. Email: [email protected] ✉ Corresponding author. Revised 2025 Jul 24; Received 2025 Apr 30; Accepted 2025 Jul 29; Issue date 2026 Apr. © 2025 The Author(s). Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. PMC Copyright notice PMCID: PMC13087836 PMID: 40916571 Abstract Anastrepha obliqua , a neotropical pest widely distributed in the Americas, attacks mango and other tropical fruits. In Mexico, it is controlled through integrated pest management, using the Sterile Insect Technique (SIT) as a main component. The applicability of SIT is significantly improved with the use of genetic sexing strains (GSS) that allow the possibility to release exclusively sterile males, the primary component of the technique. This study reports the isolation and characterization of two pupal mutations: black pupae ( bp ) and sphere pupae ( sp ), allowing for the first time the development of a genetic sexing system based on pupal color in this species. Inheritance analyses from reciprocal crosses between wild‐type and mutant individuals showed F 2 phenotypic segregation consistent with a recessive Mendelian inheritance pattern, and linkage analysis indicated that the bp and sp loci are in separate chromosomes. Using the bp mutation, two GSS were developed through gamma irradiation [T(Y; bp + )/ bp ‐22](GSS‐22) and ethyl methanesulfonate treatment [T(Y; bp + )/ bp ‐354](GSS‐354). Both GSS exhibited sex‐specific pupal differentiation but displayed a high frequency of recombinants. Despite an initial reduction in biological fitness, GSS‐22 demonstrated greater genetic stability and a lower frequency of recombinants than GSS‐354. Discrepancies between cytogenetic and genomic data, particularly regarding the localization of the gene responsible for the black pupae phenotype, underscore the need to integrate polytene chromosome and genomic analyses to characterize these translocations and improve GSS stability precisely. These results represent a breakthrough in the creation of genetic tools for the management of A. obliqua control. Keywords: chromosomal translocation, genetic sexing strain, mass rearing, recessive mutation, sterile insect technique Two spontaneous mutations in pupae of the West Indian fruit fly Anastrepha obliqua , named bp and sp , were isolated and studied. The bp mutation was selected, and using two types of Y‐autosoma l translocation inducers and an appropriate crossing scheme, two genetically sexed strains based on pupa l color were developed for the first time in this species, in which males emerge from wild‐type brown pupae and females from black pupae. One of these strains was obtained by exposure to gamma radiation and the other to methyl ethane sulfonate. Introduction Fruit flies of the genus Anastrepha Schiner (Diptera: Tephritidae) are endemic to the Americas, and include more than 250 species, distributed from North America (southern Florida, Rio Grande Valley in Texas, Mexico) to southern Argentina (Aluja, 1994 ; Hernández‐Ortiz et al. , 2012 ; Norrbom & Korytkowski, 2012 ). These include Anastrepha fraterculus (Wiedemann 1830), Anastrepha grandis (Macquart 1846), Anastrepha ludens (Loew 1873), Anastrepha obliqua (Macquart 1835), Anastrepha serpentina (Wiedemann 1830), Anastrepha striata (Schiner 1868), and Anastrepha suspensa (Loew 1862), which are considered species of economic importance, as they directly affect the mango and citrus industry and have a wide host range (Bacca et al. , 2017 ; Degracia et al. , 2023 ). A. obliqua , commonly known as the West Indian fruit fly, is one of the most polyphagous species of this genus and is considered to be the second most economically important species in Mexico (Aluja & Mangan, 2008 ; Peña et al. , 2009 ; Liedo, 2016 ). It can cause economic yield losses ranging from 25% to 70%, and the presence of adults fruit flies in commercial orchards can lead to restrictions on fruit movement in low‐prevalence or temporarily fruit‐fly‐free areas (Aluja & Birke, 1993 ; Toledo et al. , 2005 ). Area‐wide integrated pest management was first implemented in Mexico in 1992 to control A. ludens and A. obliqua , using the sterile insect technique (SIT) as the main component (Orozco‐Dávila et al. , 2017 ). The SIT is a species‐specific and environmentally safe genetic control method that involves mass‐rearing and sterilization of insects with irradiation before systematically releasing them into targeted areas to suppress, eradicate, or prevent the establishment of pest populations (Bourtzis & Vreysen, 2021 ). Some SIT applications have successfully used bisexual releases. However, the sole release of sterile males has proven to be more efficient and cost‐effective, as it maximizes matings with wild females and prevents fruit damage caused by oviposition from sterile females (Hendrichs et al. , 1995 ; Rendón et al. , 2004 ). Additionally, this approach reduces operational costs associated with rearing and release (Robinson, 2002a ). The development of genetic sexing strains (GSS) makes possible the exclusive release of males in the field, and their construction requires two genetic components: (1) Selection and isolation of recessive morphological or biochemical markers in early stages such as the embryo, larva or pupa. (2) Induction of chromosomal rearrangements such as Y‐autosomal translocation, which involves the autosome carrying the selectable marker. This approach results in pseudo‐sexual dimorphism, with all males being wild type while only females display selectable marker (Alphey, 2007 ; Franz et al. , 2021 ). Successful examples include the VIENNA 8 strain of the Mediterranean fruit fly ( Ceratitis capitata ), a quarantine pest in Mexico and a species of global economic significance (Giunti et al. , 2023 ; Aluja et al. , 2024 ). This strain utilizes the white pupae ( wp ) marker and a temperature‐sensitive lethal ( tsl ) mutation to eliminate females at the embryonic and pupal stages (Augustinos et al. , 2017 ). Other notable examples are two Anastrepha species: A. ludens and A. fraterculus , both of which use the same black pupae morphological marker ( bp ), which allowing for the separation of females with black puparia from males with brown puparia (Zepeda‐Cisneros et al. , 2014 ; Meza et al. , 2020 ). Currently, the application of SIT to A. obliqua does not allow the release of males only, a limitation that hinders operational optimization, reducing efficiency and increasing costs. To address this challenge, in 2017, the Genetics Department of the National Fruit Fly Program in Mexico launched a project to identify genetic markers in Anastrepha and develop a GSS using a classical genetic approach (Franz et al. , 2021 ). This study identified and characterized two novel genetic markers in A. obliqua and developed two GSSs using the black pupae ( bp ) visual marker. We induced chromosomal translocations by gamma irradiation and ethyl methanesulfonate (EMS) treatment, linking the autosomal wild type allele to the Y chromosome. Additionally, we assessed the biological attributes, genetic stability, and cytogenetic profiles of the resulting GSS to determine their viability and functionality in SIT application. Materials and methods Insect strain and rearing conditions This study used a wild‐type (WT) strain of A. obliqua from the mutant bank of the Programa Operativo Moscas, SADER/SENASICA‐IICA, Genetics Department in Metapa de Domiguez, Chiapas, Mexico. Flies having the black pupae ( bp ) phenotype were isolated from a mass‐reared population after screening thousands of pupae. The sphere pupae ( sp ) phenotype was isolated from a small WT colony. Wild type flies and the two mutant lines, bp and sp , were maintained under controlled conditions (70%–80% relative humidity, 26 °C, and a 12 : 12 light/dark photoperiod). Larvae were reared on an artificial diet consisting of corncob fractions (16.3%,) sugar (9.3%), yeast (7.1%), corn flour (5.4%), citric acid (0.45 %), sodium benzoate (0.41%), Nipagin (0.2%), guar gum (0.05%), and water (60.8) (Pascacio‐Villafán et al. , 2018 ). Adults were fed a 1 : 3 mixture of hydrolyzed protein and sugar, with water provided ad libitum . Genetic and fitness analysis of mutations Reciprocal single crosses between the WT strain and the possible mutant strains (♀ WT × ♂ bp , ♂ WT × ♀ bp , ♀ WT × ♂ sp and ♂ WT ×♀ sp ) were performed, to determined inheritance patterns and potential sex linkage of the mutations. The F 1 offspring were interbred in groups of five pairs, the F 2 offspring were meticulously classified using a Zeiss Stemi 2000 stereomicroscope to distinguish phenotypic variations. Potential genetic linkage between the mutations was determined by an initial cross between pure black pupae and sphere pupae individuals ( bp sp + /bp sp + × bp + sp/bp + sp ) in a single mating pair. The F 1 offspring were interbred en masse , and the F 2 offspring were classified by phenotype. To validate the type of linkage between the mutations, one F 1 male of the GSS generated in this study was evaluated with five sp mutant females. The fitness of the WT strain, homozygous bp and sp mutants, and the GSS were evaluated using five survival parameters. Egg hatching was quantified as the number of larvae emerging per 100 eggs. Five hundred eggs were incubated at 26 °C for 5 d in a KBF720 Binder bioclimatic chamber. Larval survival (LS) was determined by counting the number of larvae that reached the third instar stage. Pupal survival (PS) was measured as the percentage of larvae that pupated, calculated by dividing the total number of pupae by the total number of larvae. Adult emergence (AE) was determined as the percentage of pupae that successfully developed into adults, dividing the total number of emerged adults by the total pupae. Finally, overall fitness (OF) was calculated using the following formula: O F = L S 100 × P S 100 × A E 100 . Development of GSSs based on pupal color For the development of GSSs, bp was selected as a morphological marker due to its easy identification and its higher biological fitness compared to sp . Y‐autosome translocations were induced by gamma irradiation and ethyl methanesulfonate (EMS) treatment. In the first approach, WT pupae, 48 h before adult emergence, were exposed to doses of 5, 10, 20, 30, and 35 Gy at a dose rate of 2.7 Gy/min in a Gammabeam™‐127 (Nordion) irradiator with a cobalt 60 source. In the second approach, newly emerged adults were exposed to 10, 20, 30, and 40 mmol/L EMS via the diet for 36 h. Following treatment, WT males were crossed with homozygous bp females ( bp/bp ) at a ratio of 2000 WT males × 4000 bp/bp females per experiment. In the F 1 generation, males were selected to establish 600 independent families, each consisting of one F 1 male and 15 bp/bp females. In the F 2 generation, families in which males emerged from brown pupae (WT) and females emerged from bp were identified as potential carriers of the translocation T(Y; bp + )/ bp (Fig. 1 ). The selected families were used to develop GSS by crossing brown pupae males with black‐pupae females. In each generation, recombinants were removed (brown pupae females and black pupae males). Fig. 1. Open in a new tab Schematic representation of crosses for the development of a Genetic Sexing Strain for Anastrepha obliqua using two induction methods: gamma irradiation and EMS treatment. The approach is based on the translocation of an autosome to the Y chromosome, utilizing the black pupae mutation ( bp ) as a morphological marker. Biological attributes and genetic integrity of GSSs The biological attributes of the GSS were evaluated through mass crosses under controlled conditions. Each cross included 500 males and 500 females per strain, housed in separate 30 cm 3 cages. Fertility assessment involved collecting 500 eggs per strain, incubating them at 27 ± 1 °C in a KBF720 Binder bioclimatic chamber for 7 d, and recording the number of hatched eggs. Additionally, 500 neonate larvae per strain were transferred to the artificial diet after 4 d of incubation in aerated water. Ten days later, third‐instar larvae were counted, transferred to sawdust trays for pupation, and monitored for mature pupae and adult emergence. To assess the feasibility of maintaining the genetic integrity of both GSS, they were reared for more than ten generations using the filter rearing system (FRS) (Fisher & Cáceres, 2000 ). This involved removing males that emerged from black pupae and females that emerged from brown pupae. Thus, each generation underwent genetic filtering to eliminate recombinants, and maximum egg hatch was evaluated in triplicate, with 100 eggs per replicate per generation. Recombination rate and sex ratio were recorded in each generation. Cytogenetic analysis Mitotic chromosomes were obtained from the third‐instar larval brains of the F 8 generation of GSS‐selected flies, following the protocol described by García‐Martínez et al. ( 2009 ). Brain tissue was dissected in a 1% sodium citrate hypotonic solution and incubated for 15 min. Afterward, the tissue was fixed in a freshly prepared 3 : 1 methanol‐acetic acid solution for 5 min. Cells were macerated on clean slides using 60% acetic acid on a 45 °C hot plate until thoroughly dried. Chromosomes were stained for 2 h in a 10% Giemsa solution prepared in sodium phosphate buffer. Microscopic analysis was conducted at 100× magnification using a Carl Zeiss Axioskop 40 microscope, and images were acquired with an AxioCam HRm camera. Only well‐spread metaphases with clearly paired homologous metaphase chromosomes were analyzed. Chromosome length was measured using ImageJ software and expressed as a percentage of the total diploid complement length. Data analysis F 2 phenotypic frequencies were analyzed using contingency tables and Pearson's chi‐square tests to determine the inheritance pattern of bp and sp mutations. The fitness of WT and homozygous mutants ( bp and sp ) was evaluated using two statistical approaches, depending on normality and variance homogeneity. When data followed a normal distribution and showed homogeneous variances, ANOVA was applied, followed by Tukey's post hoc test to identify specific differences. The Kruskal–Wallis test was used for non‐normal data, followed by Dunn's post hoc test with Bonferroni correction ( P < 0.05). This approach provided a robust and comprehensive statistical framework for evaluating the variables. The Shapiro–Wilk test was used to assess normality for each variable in both GSS's, and Levene's test was applied to verify the homogeneity of variances. When both assumptions were met ( P > 0.05), a Student's t ‐test assuming equal variances was used to compare means between strains. For overall fitness, where the normality assumption was not satisfied for T(Y; bp + )/ bp ‐22 (see below), the non‐parametric Mann–Whitney U test was applied. Statistical significance was considered at P ≤ 0.05. All statistical analyses were conducted in R (v4.4.0) using the tidyverse, car, and rstatix packages. Results Morphological description and genetic analysis of the bp and sp mutants We identified and characterized two novel mutations: black pupae ( bp ) and sphere pupae ( sp ), displaying distinct morphological differences. The bp mutation has black puparian color, in contrast to the typical wild type reddish‐brown coloration of A. obliqua (Fig. 2A ). Additionally, bp adults exhibit darker thorax, abdomen, and wings than those of the wild‐type strain (Fig. 2B ). This phenotype is also evident in the larval stage, as the anal lobes display dark pigmentation. In contrast, the sp mutation results in a spherical pupal shape. Adults have a shorter thorax and abdomen than WT individuals, and both share the same normal coloration (Fig. 2C ). Fig. 2. Open in a new tab Phenotype of pupae and adult males and females of the wild‐type strain (A) compared to the mutant strains black pupae (B) and sphere pupae (C). Genetic and fitness analysis of mutations F 1 offspring of the four reciprocal crosses between WT and mutant flies displayed a WT phenotype. Inbreeding of the F 1 progeny in each case resulted in an F 2 offspring that exhibited both WT and mutant phenotypes. The F 2 phenotypic frequencies confirmed that phenotype segregation followed the expected pattern for a recessive Mendelian inheritance, showing no significant deviations from a theoretical 3 : 1 WT to mutant ratio (Table 1 ). This indicated that the inheritance of each mutation is controlled individually by a single autosomal recessive gene. On the other hand, the original cross between the bp and sp mutant produced only WT offspring in F 1 . This F 1 offspring was interbred in mass and the F 2 offspring showed a ratio close to 9 : 3 : 3 : 1 WT to bp to sp to bp sp , indicating that segregation was independent, and that the two loci are on separate chromosomes (Table 1 ). Backcrossing between a male of T(Y; bp + )/ bp ‐22 (see below) and sp females, did not show sexing for the sp marker, confirming that these two loci ( bp and sp ) are not linked (Table 1 ). Table 1. Inheritance pattern and genetic linkage analysis of black pupae and sphere pupae mutations Inheritance crosses (♂ × ♀) Phenotype of F 2 offspring No. of cages WT bp sp bp sp χ 2 bp × WT 4 4837 1525 3.60 † WT × bp 4 5953 1919 1.63 † sp × WT 6 946 282 2.71 † WT × sp 6 2728 853 2.66 † Linkage cross sp × bp 1 225 75 78 23 0.27 ‡ Backcross GSS‐22 F 1 × sp 1 ♂ 35 ♀ 46 ♂ 26 ♀ 18 Open in a new tab † Hypothesis 3 : 1; χ 2 0.05, df = 1 < 3.841. ‡ Hypothesis 9 : 3 : 3 : 1; χ 2 0.05, df = 3 < 7.82. The evaluation of biological attributes revealed fitness differences between the WT strain and the bp and sp mutants, showing significant variation in larval survival ( F = 8.063, P = 0.0018), pupal survival ( χ 2 = 7.51, P = 0.0233), adult emergence ( χ 2 = 18.60, P < 0.001), and overall fitness ( F = 25.19, P < 0.001). However, fertility among the three strains showed no significant differences ( F = 2.158, P = 0.128). The egg‐to‐larval survival of the bp was significantly lower than WT ( P = 0.0013), while bp and sp showed no significant differences. Larva to pupal survival differed significantly between bp and sp ( P = 0.0348) but remained similar between WT and the two mutants. For pupal to adult survival (adult emergence), sp exhibited significantly lower values than bp ( P = 0.0005) and WT ( P = 0.0007). Finally, overall fitness was significantly lower in bp ( P = 0.0003) and sp ( P < 0.0001) compared to WT, with sp displaying the lowest overall fitness relative to bp ( P = 0.0414) (Table 2 ). Table 2. Percentage values for fitness and survival measures of the black pupae ( bp ) and sphere pupae ( sp ) mutations compared to the wild‐type strain (WT) Strain Fertility Egg to larvae survival Larva to pupae survival Pupa to adults survival Overall fitness WT 87.93 ± 0.530 a 66.90 ± 4.715 a 97.12 ± 0.862 ab 86.42 ± 1.488 a 56.3 ± 4.315 a bp 87.33 ± 3.028 a 36.10 ± 4.983 b 97.58 ± 0.928 a 86.24 ± 2.238 a 30.3 ± 4.320 b sp 89.40 ± 3.202 a 47.90 ± 6.541 ab 93.18 ± 1.233 b 36.31 ± 7.361 b 15.3 ± 3.735 c Open in a new tab Values represent mean ± standard error, and different letters within a column indicate statistically significant differences between strains ( P ≤ 0.05). Development of GSSs using the bp marker Following a crossing and backcrossing scheme (Fig. 1 ), a total of 17 400 F 1 males were analyzed using the gamma irradiation induction method, and 12 000 F 1 males using the EMS induction method. In both induction methods, the highest doses generated Y‐linked translocated lines. Using a 35 Gy irradiation dose, we identified a strain designated as T(Y; bp + )/ bp ‐22 (GSS‐22). In contrast, a 40 mmol/L EMS dose produced the strain T(Y; bp + )/ bp ‐354 (GSS‐354). No Y‐autosome translocations were recovered at lower irradiation doses (5, 10, 20, or 30 Gy) or at lower EMS concentrations (10, 20, or 30 mmol/L). Both strains exhibited the same phenotypic pattern: males emerged from brown pupae (WT), whereas females emerged from black pupae. However, a proportion of recombinant males was present. Both strains remain under continuous rearing using a generational FRS, with systematic evaluations of their phenotype and fertility parameters. Biological attributes and genetic integrity of GSSs No statistically significant differences were found between the GSS‐22 and GSS‐364 strains for any of the biological attributes evaluated. Student's t ‐test results were as follows: egg hatching ( t = 1.92, P = 0.090), larval survival ( t = 1.26, P = 0.243), pupal survival ( t = −1.41, P = 0.195), and adult emergence ( t = 0.67, P = 0.521). Also, there were no significant differences in overall fitness between strains ( W = 17, P = 0.345). Nevertheless, a consistent pattern was observed across the biological parameters. Fertility and larval survival showed the lowest values in both strains, with mean egg hatching percentages ranging from 17.8% to 19.8%, and larval survival between 23.8% and 26.0%. In contrast, both egg‐to‐pupa and pupa‐to‐adult survival rates exceeded 90%, indicating high performance in the later developmental stages. The overall fitness remained low in both strains, with average values around 4%, and no significant differences were detected (Table 3 ). Table 3. Percentage values for survival measures and fitness across the genetic sexing strains, GSS‐22 and GSS‐364 Strain Fertility Larvae survival Egg to pupa survival Pupa to adult survival Overall fitness GSS‐22 19.20 ± 1.30 a 26.00 ± 3.08 a 91.37 ± 3.33 a 93.68 ± 5.18 a 4.4 ± 0.40 a GSS‐364 17.80 ± 2.17 a 23.80 ± 2.39 a 94.02 ± 2.54 a 91.31 ± 5.96 a 3.4 ± 0.37 a Open in a new tab Values represent mean ± standard error, and different letters within a column indicate statistically significant differences between strains ( P ≤ 0.05). To ensure genetic integrity, an FRS was applied to the GSS‐22 and GSS‐364 strains for 14 and 11 generations, respectively. The GSS‐22 parental cross began with 25 males and 35 females, and the number progressively increased to 1850 males and 1740 females by F 14 (Table 4 ). The GSS‐364 parental cross began with 15 males and 18 females, by F 11 , it had grown to 1449 males and 1392 females (Table 5 ). Population growth in both GSS correlated with the low hatch rates observed in early generations. Fertility increased in both GSS lines across generations, reaching average maximum egg hatch rates of 23.33% for GSS‐22 and 20.67% for GSS‐364. Compared to the F 1 , both GSS showed significant increases in fertility. GSS‐22 rose from 5% in F 1 to 23.33% in F 14 (Table 4 ), while GSS‐364 exhibited a more moderate increase, from 14.33% in F 1 to 22% in F 11 (Table 5 ). Table 4. Phenotypes, fertility and recombination obtained from filter rearing system applied to GSS‐22 obtained through radiation (35 Grays) Generation Brown pupae Brown pupae adults Black pupae Black pupae adults Fertility (mean ± SD) (%) Recombinants Brown pupae (%) Recombinants Black pupae (%) ♂ ♀ ♂ ♀ Parental 25 25 0 38 7 31 4.33 ± 1.53 0.00 11.11 F 1 24 23 0 49 5 43 5.00 ± 1.00 0.00 7.04 F 2 125 117 0 159 44 94 9.33 ± 1.15 0.00 17.25 F 3 259 204 0 281 58 175 8.33 ± 1.53 0.00 13.27 F 4 258 245 0 285 78 204 10.00 ± 2.00 0.00 14.80 F 5 247 240 0 299 40 245 11.67 ± 2.08 0.00 7.62 F 6 217 196 0 275 54 207 13.33 ± 2.31 0.00 11.82 F 7 350 347 0 393 77 299 16.00 ± 1.73 0.00 10.65 F 8 390 375 0 428 53 375 15.33 ± 1.15 0.00 6.60 F 9 503 478 0 625 82 543 20.67 ± 3.06 0.00 7.43 F 10 1002 973 0 1189 221 875 20.67 ± 3.61 0.00 10.68 F 11 829 817 0 957 102 839 21.33 ± 2.52 0.00 5.80 F 12 1253 1240 0 1429 295 1125 21.67 ± 3.06 0.00 11.09 F 13 1425 1403 2 1687 235 1411 23.00 ± 2.00 0.07 7.71 F 14 1865 1850 3 2068 328 1740 23.33 ± 0.58 0.08 8.37 Open in a new tab Table 5. Phenotypes, fertility, and recombination obtained from the filter rearing system applied to GSS‐364 obtained through EMS (40 mmol/L) Generation Brown pupae Brown pupae adults Black pupae Black pupae adults Fertility (mean ± SD) (%) Recombinants Brown pupae (%) Recombinants Black pupae (%) ♂ ♀ ♂ ♀ Parental 18 15 0 23 3 18 13 ± 2.65 0.00 8.33 F 1 32 31 0 62 9 41 14.33 ± 3.21 0.00 11.11 F 2 35 31 0 42 5 35 10.30 ± 1.53 0.00 7.04 F 3 85 77 1 92 6 84 22.33 ± 3.51 0.60 3.59 F 4 112 105 0 128 12 110 20.67 ± 4.51 0.00 5.29 F 5 120 115 0 153 14 139 16.00 ± 2.00 0.00 5.22 F 6 540 523 4 745 89 629 17.00 ± 1.73 0.32 7.17 F 7 825 798 10 983 245 629 21.67 ± 3.06 0.59 14.65 F 8 832 790 35 968 163 697 19.33 ± 1.53 5.82 9.88 F 9 1025 760 148 1325 320 925 21.67 ± 1.15 6.87 15.96 F 10 1324 1197 123 1425 247 1489 22.33 ± 3.06 4.02 8.42 F 11 1512 1449 53 1759 367 1392 22.00 ± 2.00 1.63 11.44 Open in a new tab In GSS‐22, the male recombination frequency fluctuated between 5.80% and 17.25% across generations, while recombinant females did not appear until F 13 , when the population was scaled to 1000 pairs per cage, reaching 0.065% and slightly increasing to 0.077% in F 14 (Table 4 ). In contrast, GSS‐364 displayed both male and female recombinants from early generations. The male recombination frequency ranged from 3.59% to 15.96%, while female recombinants reached up to 6.87% in F 9 (Table 5 ). These results suggest that the integrity of genetic can be maintained by the application of an FRS in the GSS‐22 better than GSS‐364, over the generations evaluated. Cytogenetic analysis Since GSS‐22 showed the greatest stability in integrity across generations using FRS, it was selected for cytogenetic analysis, and 300 metaphase spreads of mitotic chromosomes were prepared. We selected 39 with well‐extended chromosomes paired with their homologs to ensure the highest measurement accuracy. Among these, 28 (71.7%) exhibited differences between homologs of chromosome pair 3 (Fig. 3 ). The X chromosome and chromosome 2, recognizable by their distinctive length and morphology, served as references to validate the analysis results. However, cytogenetic analysis also revealed chromosomal breakage in other regions (Fig. 3 ) and less frequent alterations in other autosomes. In total, we identified modifications in chromosome pair 4 (7 metaphases; 17.9%), chromosome 5 (4 metaphases; 10.2%), and chromosome 6 (1 metaphase; 2.5%) (Table 6 ). Fig. 3. Open in a new tab Cytogenetic representation of (A) mitotic metaphase chromosomes from the brain ganglia of a third‐instar male larva of the wild‐type A. obliqua strain and (B) mitotic metaphase chromosomes of the GSS‐22. The three arrowheads highlight chromosomal regions that differ from the wild‐type complement. Table 6. Comparison of chromosome length and relative differences between the wild‐type strain and the translocated strain GSS‐22 CR Wild type T(Y/ bp + )‐22 TL (µm) DL (µm) LR (%) DRL (%) TL (µm) DL (µm) LR (%) DRL (%) 2 12.64 ± 2.27 0.27 ± 0.18 11.32 ± 0.73 0.24 ± 0.14 11.68 ± 2.17 0.13 ± 0.58 11.20 ± 0.72 0.12 ± 0.09 12.37 ± 2.66 11.08 ± 0.74 11.55 ± 2.16 11.08 ± 0.71 3 9.70 ± 2.12 0.21 ± 0.16 8.67 ± 0.46 0.19 ± 0.13 9.35 ± 1.62 1.14 ± 0.89 8.98 ± 0.42 1.07 ± 0.78 9.49 ± 2.02 8.48 ± 0.49 8.22 ± 1.49 † 7.91 ± 0.81 4 9.23 ± 2.01 0.17 ± 0.13 8.26 ± 0.29 0.15 ± 0.09 8.74 ± 1.59 0.26 ± 0.35 8.38 ± 0.35 0.25 ± 0.30 9.07 ± 1.95 8.11 ± 0.30 8.48 ± 1.59 8.13 ± 0.51 5 8.80 ± 1.98 0.18 ± 0.11 7.86 ± 0.38 0.17 ± 0.11 8.48 ± 1.62 0.34 ± 0.57 7.91 ± 0.35 0.32 ± 0.49 8.62 ± 1.97 7.70 ± 0.39 7.91 ± 1.45 7.59 ± 0.49 6 8.14 ± 1.66 0.14 ± 0.11 7.31 ± 0.39 0.13 ± 0.11 7.71 ± 1.46 0.21 ± 0.29 7.39 ± 0.44 0.20 ± 0.27 8.00 ± 1.69 7.18 ± 0.40 7.51 ± 1.45 7.19 ± 0.50 X 8.63 ± 1.75 ‒ 7.75 ± 0.75 ‒ 8.02 ± 1.18 ‒ 7.77 ± 0.78 ‒ Y 6.99 ± 1.44 ‒ 6.29 ± 0.61 6.65 ± 0.92 † 6.45 ± 0.59 Open in a new tab CR, chromosome; TL, total length; DL, difference in length; LR, relative length; DRL, difference in relative length. † Possibly involved in translocation. Discussion In this study, two pupal mutations of A. obliqua that affect the color ( black pupae , bp ) and shape ( sphere pupa , sp ) of the puparium were isolated and characterized. GSSs were constructed for the first time for this pest species. The bp mutation showed a phenotype and inheritance similar to the black pupae mutation of A. ludens and A. fraterculus (Zepeda‐Cisneros et al. , 2014 ; Meza et al. , 2020 ), which is an efficient visual marker for sex differentiation compared to sp , whose phenotypic detection is more challenging under mass‐rearing conditions (Rössler, 1979). Both mutations, bp and sp , follow a recessive Mendelian inheritance pattern, aligning with the expected phenotypic ratios (Ward et al. , 2021 ; Paulo et al. , 2025 ). Linkage analysis indicated that the genes responsible for these mutations are on different chromosomes. Biological performance analyses revealed that both mutations significantly affect individual fitness. The bp mutation reduces larval survival compared to the WT strain, while sp shows the lowest fitness among all evaluated lines. These results align with the pleiotropic effects associated with these mutations, which, in addition to altering the phenotype, may interfere with various physiological and behavioral processes (Takahashi, 2013 ; Meza et al. , 2019 ). We selected the bp mutation for the development of a GSS, which enabled the construction of the GSS‐22 and GSS‐354 through irradiation (35 Gy) and EMS treatment (40 mmol/L), respectively. Both methodologies generated functional lines, although they initially showed reductions in biological parameters and fitness compared to the WT and bp lines. These reductions are characteristic of translocated lines and are consistent with previous studies in other tephritid species (Cáceres et al. , 2004 ; Vreysen et al. , 2021 ). Although precise thresholds for optimal irradiation remain undefined, studies on A. obliqua have shown that doses ≥ 25 Gy induce high sterility, while doses ≥ 60 Gy markedly reduce viability with 98% sterility and disrupt behavior (Toledo et al. , 2004 ; Gallardo‐Ortiz et al. , 2018 ). Consistent with these findings, exposure of pupae to 35 Gy induced the targeted Y‐autosome translocation but yielded very low F 1 egg‐hatch rates, 4.0% ± 0.5% in GSS‐22 versus 13.2% ± 1.1% in GSS‐364 (EMS) (Meza et al. , 2019 ). Translocation‐based GSS are inherently semi‐sterile, under Mendelian segregation, only half of the gametes inherit the balanced Y‐autosome complement required for viability (Robinson, 1976 ), and pleiotropic or off‐target effects often reduce hatch rates. For example, male‐linked translocation in Culex pipiens maintains egg‐hatch rates of only ∼15% (Laven & Aslamkhan, 1970 ; Robinson, 1976 ). Despite this initial suppression, both lines demonstrated progressive increases in egg‐hatch rates, rising from 5.0% (F 1 ) to 23.3% (F 14 ) in GSS‐22, and from 14.3% (F 1 ) to 22.0% (F 11 ) in GSS‐364. To our knowledge, no prior Tephritidae GSS study has reported such detailed, generation‐by‐generation hatch data documenting fertility recovery. These findings support the hypothesis that homogeneous rearing combined with a filter rearing system gradually eliminates unbalanced chromosomal rearrangements and mitigates the fitness costs inherent to Y‐autosome translocations (Rössler, 1979 ; Toledo et al. , 2004 ; Zepeda‐Cisneros et al. , 2014 ; Franz, 2001 ). Beyond fertility, the two lines diverged in genetic stability. The irradiation‐induced GSS‐22 exhibited greater long‐term genetic stability, characterized by a progressive decline in male recombinant frequency and the emergence of female recombinants in generations F 13 (0.065%) and F 14 (0.077%). This result may be attributed to the effects of double‐strand breaks induced by irradiation, which facilitate the formation of targeted chromosomal rearrangements (Robinson, 1976 ; Robinson, 2002b ). In contrast, the EMS‐derived GSS‐364 line displayed lower genetic stability, marked by a higher frequency of male recombinants in early generations and a notable increase in female recombinants starting in F 7 . This phenomenon may stem from EMS's mode of action, which induces point mutations by alkylating nitrogenous bases and causing genomic alterations. Thus, unlike irradiation, EMS leads to the accumulation of recessive lethal and sub‐lethal alleles that are unmasked under successive inbreeding and at higher doses, can also provoke small insertions/deletions and local chromosomal lesions, further undermining the production of balanced gametes (Ohnishi, 1977 ; Sega, 1984 ; Kodym & Afza, 2003 ; Chen et al. , 2023 ). The black pupae phenotype results from mutations in ebony , a key gene in melanin synthesis that regulates the conversion of dopamine into N‐β‐alanyl dopamine (NBAD), the precursor of yellow sclerotin in the cuticle. Mutations in ebony disrupt this pathway, redirecting dopamine toward melanin production and resulting in individuals with hyperpigmented cuticles (Paulo et al. , 2025 ). The high conservation of ebony across different Anastrepha species suggests that there is strong evolutionary pressure to maintain its function. In A. ludens , this gene has been mapped to mitotic chromosome 2 (Paulo et al. , 2025 ), whereas in A. obliqua , the genomic assembly GCF_027943255.1 places it on chromosome 1 ( XM_054870789.1 ). This chromosome appears homologous to chromosome 2 in A. ludens , exhibiting a similar chromosomal localization in both species. Despite this evidence, cytogenetic analysis of the GSS‐22 revealed that the predominant translocation involves chromosome 3, given the high frequency of alterations in its length. However, detecting breaks in other chromosomes suggests a more complex chromosomal arrangement. Although the analyzed metaphases did not show frequent morphological differences in other autosomes, the possibility of undetected rearrangements through mitotic chromosome analysis highlights the limitations of this approach for characterizing chromosomal translocations. This limitation is particularly relevant in species like A. obliqua , whose autosomes exhibit similar morphology (Ibáñez‐Palacios et al. , 2010 ; Zepeda‐Cisneros et al. , 2014 ). Previous studies on tephritids have shown that accurately characterizing translocations requires complementary polytene chromosome analysis (Zacharopoulou et al. , 2017 ). Integrating this approach with genomic analyses would improve the resolution of chromosomal rearrangements in GSS‐22 and help clarify discrepancies observed in mitotic analyses. Additionally, sequential back‐crossing of bp –Y translocated males with bp females, coupled with stringent selection for high male fertility, should mitigate irradiation‐induced genomic instability by accelerating the loss of secondary chromosomal rearrangements unlinked to the breakpoint while preserving the translocation (Hospital, 2005 ; Isasawin et al. , 2014 ; Ntoyi et al. , 2022 ). This study provides the first report of the bp and sp mutations in A. obliqua and their application in GSS development. GSS‐22, generated through irradiation, exhibited greater genetic stability and functionality than GSS‐364, which resulted from EMS treatment. Discrepancies between cytogenetic and genomic data underscore the need for integrated strategies to resolve the chromosomal structure of GSS‐22. Our immediate aims are to enhance fertility through directed selection and to conduct semi‐mass‐rearing trials that evaluate key quality‐control metrics alongside genetic stability, utilizing optimized filter‐rearing protocols to minimize female recombinants. This study represents a significant advance in A. obliqua genetic control and the potential for their implementation in area‐wide sterile‐insect technique programs. Disclosure All the authors confirm there is no conflict of interest. Acknowledgments We thank Al Handler for reviewing an earlier version of this manuscript. We thank the technicians of the Genetics Department Hilda García Mendoza and Daniel López Gómez. We also thank Angel Humberto de León Crisóstomo, who participated in searching for the mutation in the mass rearing and establishing the colony. This study was supported by the International Atomic Energy research contract no. 23469 as part of the Coordinated Research Project “Generic Approach for the Development of Genetic Sexing Strains for SIT Applications” (CRP code D44003). References Alphey, L.S. (2007) Engineering insects for the sterile insect technique. In Area‐wide Control of Insect Pests: From Research to Field Implementation (eds. Vreysen M.J.B., Robinson A.S. & Hendrichs J.), pp. 51–60. Springer; Netherlands, Dordrecht. [ Google Scholar ] Aluja, M. and Birke, A. (1993) Habitat use by adults of Anastrepha obliqua (Diptera: Tephritidae) in a mixed mango and tropical plum orchard. Annals of the Entomological Society of America, 86, 799–812. [ Google Scholar ] Aluja, M. (1994) Bionomics and management of Anastrepha . Annual Review of Entomology, 39, 155–178. [ Google Scholar ] Aluja, M. , Guillén, L. , Pascacio‐Villafán, C. , Juárez‐Durán, M. , Miranda‐Salcedo, M.A. and Liedo, P. (2024) Management of economically important native and exotic fruit fly (Tephritidae) species in Mexico. In Management of Fruit Flies in the Americas (ed. Mello Garcia F.R.), pp. 355–406. Springer, Cham. [ Google Scholar ] Aluja, M. and Mangan, R.L. (2008) Fruit fly (Diptera: Tephritidae) host status determination: critical conceptual, methodological, and regulatory considerations. Annual Review of Entomology, 53, 473–502. [ DOI ] [ PubMed ] [ Google Scholar ] Augustinos, A.A. , Targovska, A. , Cancio Martinez, E.I. , Schorn, E. , Franz, G. , Cáceres, C. et al . (2017) Ceratitis capitata genetic sexing strains: laboratory evaluation of strains from mass‐rearing facilities worldwide. Entomologia Experimentalis et Applicata, 164, 305–317. [ Google Scholar ] Bacca, T. , Canal, N.A. and Cruz, M.I. (2017) Diversidad de las moscas de las frutas (Diptera: Tephritidae) y sus parasitoides en siete municipios del departamento de Nariño. Boletín Científico. Centro De Museos. Museo De Historia Natural, 21, 81–98. [ Google Scholar ] Bourtzis, K. and Vreysen, M.J.B. (2021) Sterile insect technique (SIT) and its applications. Insects, 12, 638. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Cáceres, C. , Cayol, J.P. , Enkerlin, W.R. , Franz, G. , Hendrichs, J. and Robinson, A.S. (2004) Comparison of Mediterranean fruit fly ( Ceratitis capitata ) (Tephritidae) bisexual and genetic sexing strains: development, evaluation and economics. In Proceedings, Symposium: 6th International Symposium on Fruit Flies of Economic Importance (ed. Barnes, B.N. ), 6–10 May 2002, Stellenbosch, South Africa, pp. 367–381. Isteg Scientific Publications, Irene, South Africa. [ Google Scholar ] Chen, L. , Duan, L. , Sun, M. , Yang, Z. , Li, H. , Hu, K. et al . (2023) Current trends and insights on EMS mutagenesis application to studies on plant abiotic stress tolerance and development. Frontiers in Plant Science, 13, 2022. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Degracia, A.B. , Jiménez, J.Á. , Alvarado, A.B. , Valdespino, R.A. and Altamiranda‐Saavedra, M. (2023) Evaluation of the effect of the ENSO cycle on the distribution potential of the genus Anastrepha of horticultural importance in the Neotropics and Panama. Insects, 14, 714. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Fisher, K. and Cáceres, C. (2000) A filter rearing system for mass reared genetic sexing strains of Mediterranean fruit fly (Diptera: Tephritidae). In Area‐Wide Control of Fruit Flies and Other Insect Pests: Joint Proceedings of the International Conference in Area‐Wide Control of Insects Pests (ed. Tan K.H.), May 28–June 2 1998; and the Fifth International Symposium on Fruit Flies of Economic Importance, June 1–5 1998, Penang, Malaysia, pp. 543–550. Universiti Sains, Pulau Pinang, Malaysia. [ Google Scholar ] Franz, G. (2001) The genetic basis of SIT and all‐male strains pp. 25‐47. In Sterile insect technique as an environmentally friendly and effective insect control system: Proceeding of a seminar, Funchal, Madeira, Portugal, 12 ‐ 13 November 1999 , Madeira Regional Direction of Agriculture, Portugal. Franz, G. , Bourtzis, K. and Cáceres, C. (2021) Practical and operational genetic sexing systems based on classical genetic approaches in fruit flies, an example for other species amenable to large‐scale rearing for the sterile insect technique. In Sterile Insect Technique: Principles and Practice in Area‐Wide Integrated Pest Management (eds. Dyck V.A., Hendrichs J. & Robinson A.S.), 2nd edn, pp. 575–604. CRC Press, Boca Raton. [ Google Scholar ] Gallardo‐Ortiz, U. , Pérez‐Staples, D. , Liedo, P. , and Toledo, J. (2018) Sexual competitiveness, field survival, and dispersal of Anastrepha obliqua (Diptera: Tephritidae) fruit flies irradiated at different doses. Journal of Economic Entomology, 111, 761–769. [ DOI ] [ PubMed ] [ Google Scholar ] García‐Martínez, V. , Hernández, E. , Zepeda‐Cisneros, C.S. , Robinson, A.S. , Zacharopoulou, A. and Franz, G. (2009) Mitotic and polytene chromosome analysis in the Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae). Genome, 52, 20–30. [ DOI ] [ PubMed ] [ Google Scholar ] Giunti, G. , Benelli, G. , Campolo, O. , Canale, A. , Kapranas, A. , Liedo, P. et al . (2023) Biology, ecology and invasiveness of the Mediterranean fruit fly, Ceratitis capitata : a review. Entomologia Generalis, 43, 1221–1239. [ Google Scholar ] Hendrichs, J. , Franz, J. and Rendon, P. (1995) Increased effectiveness and applicability of the sterile insect technique through male‐only releases for control of Mediterranean fruit flies during fruiting seasons. Journal of Applied Entomology, 119, 371–377. [ Google Scholar ] Hernández‐Ortiz, V. , Bartolucci, A.F. , Morales‐Valles, P. , Frías, D. and Selivon, D. (2012) Cryptic species of the Anastrepha fraterculus complex (Diptera: Tephritidae): a multivariate approach for the recognition of South American morphotypes. Annals of the Entomological Society of America, 105, 305–318. [ Google Scholar ] Hospital, F. (2005) Selection in backcross programmes. Philosophical Transactions of the Royal Society B: Biological Sciences, 360, 1503–1511. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ibañez‐Palacios, J. , García‐Velazquez, A. , Zepeda‐Cisneros, C.S. and Corona‐Torres, T. (2010) Análisis cariotípico y diferenciación de cromosomas sexuales en cuatro especies de Anastrepha (Diptera: Tephritidae). Agrociencia, 44, 691–700. [ Google Scholar ] Isasawin, S. , Aketarawong, N. , Lertsiri, S. and Thanaphum, S. (2014) Development of a genetic sexing strain in Bactrocera carambolae (Diptera: Tephritidae) by introgression of sex sorting components from B. dorsalis , Salaya1 strain. BMC Genomic Data, 15, S2. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Kodym, A. and Afza, R. (2003) Physical and chemical mutagenesis. In Methods in Molecular Biology . Plant Functional Genomics (ed. Grotewold E.), pp. 189–204. Humana Press. [ DOI ] [ PubMed ] [ Google Scholar ] Laven, H. and Aslamkhan, M. (1970) Control of Culex pipiens pipiens and C. p. fatigans with integrated genetical systems. Pakistan Journal of Science, 22, 303–312. [ Google Scholar ] Liedo, P. (2016) Management of fruit flies in Mexico. In Fruit Fly Research and Development in Africa—Towards a Sustainable Management Strategy to Improve Horticulture (eds. Ekesi S., Mohamed S. & De Meyer M.), pp. 695–704. Springer, Cham. [ Google Scholar ] Meza, J.S. , Cáceres, C. and Bourtzis, K. (2019) Slow larvae mutant and its potential to improve the pupal color‐based genetic sexing system in Mexican fruit fly (Diptera: Tephritidae). Journal of Economic Entomology, 112, 1604–1610. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Meza, J.S. , Bourtzis, K. , Zacharopoulou, A. , Gariou‐Papalexiou, A. and Cáceres, C. (2020) Development and characterization of a pupal‐colour based genetic sexing strain of Anastrepha fraterculus sp. 1 (Diptera: Tephritidae). BMC Genetics, 21, 134. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Norrbom, A.L. and Korytkowski, C.A. (2012) New species of Anastrepha (Diptera: Tephritidae), with a key for the species of the megacantha clade. Zootaxa, 3478, 510–552. [ Google Scholar ] Ntoyi, N.L. , Mashatola, T. , Bouyer, J. , Kraupa, C. , Maiga, H. , Mamai, W. et al . (2022) Life‐history traits of a fluorescent Anopheles arabiensis genetic sexing strain introgressed into South African genomic background. Malaria Journal, 21, 254. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ohnishi, O. (1977) Spontaneous and ethyl methanesulfonate‐induced mutations controlling viability in Drosophila melanogaster . II. Homozygous effect of polygenic mutations. Genetics, 87, 529–545. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Orozco‐Dávila, D. , Quintero, L. , Hernández, E. , Solís, E. , Artiaga, T. , Hernández, R. et al . (2017) Mass rearing and sterile insect releases for the control of Anastrepha spp. pests in Mexico— a review. Entomologia Experimentalis et Applicata, 164, 176–187. [ Google Scholar ] Pascacio‐Villafán, C. , Guillén, L. , Williams, T. and Aluja, M. (2018) Effects of larval density and support substrate in liquid diet on productivity and quality of artificially reared Anastrepha ludens (Diptera: Tephritidae). Journal of Economic Entomology, 111, 2281–2287. [ DOI ] [ PubMed ] [ Google Scholar ] Paulo, D.F. , Nguyen, T.N. , Ward, C.M. , Corpuz, R.L. , Kauwe, A.N. , Rendon, P. et al . (2025) Functional genomics implicates ebony in the black pupae phenotype of tephritid fruit flies. Communications Biology, 8, 60. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Peña, J.E. , Aluja, M. and Wysoki, M. (2009) Pests. In The Mango: Botany, Production and Uses (ed. Litz R.E.), pp. 317–366. CAB International, Wallingford, UK. [ Google Scholar ] Povirk, L.F. (2006) Biochemical mechanisms of chromosomal translocations resulting from DNA double‐strand breaks. DNA Repair, 5, 1199–1212. [ DOI ] [ PubMed ] [ Google Scholar ] Rendón, P. , McInnis, D. , Lance, D. and Stewart, J. (2004) Medfly (Diptera: Tephritidae) genetic sexing: large‐scale field comparison of males‐only and bisexual sterile fly releases in Guatemala. Journal of Economic Entomology, 97, 1547–1553. [ DOI ] [ PubMed ] [ Google Scholar ] Robinson, A.S. (1976) Progress in the use of chromosomal translocations for the control of insect pests. Biological Reviews of the Cambridge Philosophical Society, 51, 1–24. [ DOI ] [ PubMed ] [ Google Scholar ] Robinson, A.S. (2002a) Genetic sexing strains in medfly, Ceratitis capitata , sterile insect technique programmes. Genetica, 116, 5–13. [ DOI ] [ PubMed ] [ Google Scholar ] Robinson, A.S. (2002b) Mutations and their use in insect control. Mutation Research/Reviews in Mutation Research, 511, 113–132. [ DOI ] [ PubMed ] [ Google Scholar ] Rössler, Y. (1979) Automated sexing of Ceratitis capitata (Dip.: Tephritidae): the development of strains with inherited, sex‐limited pupal color dimorphism. Entomophaga, 24, 411–416. [ Google Scholar ] Sega, G.A. (1984) A review of the genetic effects of ethyl methanesulfonate. Mutation Research/Reviews in Genetic Toxicology, 134, 113–142. [ DOI ] [ PubMed ] [ Google Scholar ] Takahashi, A. (2013) Pigmentation and behavior: potential association through pleiotropic genes in Drosophila . Genes, Genetics and Systems, 88, 165–174. [ DOI ] [ PubMed ] [ Google Scholar ] Toledo, J. , Rull, J. , Oropeza, A. , Hernández, E. and Liedo, P. (2004) Irradiation of Anastrepha obliqua (Diptera: Tephritidae) revisited: optimizing sterility induction. Journal of Economic Entomology, 97, 383–389. [ DOI ] [ PubMed ] [ Google Scholar ] Toledo, J. , Perez, C. , Liedo, P. and Ibarra, J. (2005) Susceptibilidad de larvas de Anastrepha obliqua Macquart (Diptera: Tephritidae) a Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae) en condiciones de laboratorio. Vedalia, 12, 11–22. [ Google Scholar ] Vreysen, M.J.B. , Abd‐Alla, A.M.M. , Bourtzis, K. , Bouyer, J. , Caceres, C. , De Beer, C. et al . (2021) The Insect Pest Control Laboratory of the Joint FAO/IAEA Programme: ten years (2010–2020) of research and development, achievements and challenges in support of the sterile insect technique. Insects, 12, 346. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Ward, C.M. , Aumann, R.A. , Whitehead, M.A. , Nikolouli, K. , Leveque, G. , Gouvi, G. et al . (2021) White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter. Nature Communications, 12, 491. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Zacharopoulou, A. , Augustinos, A. , Drosopoulou, E. , Tsoumani, K. , Gariou‐Papalexiou, A. , Franz, G. et al . (2017) A review of more than 30 years of cytogenetic studies of Tephritidae in support of sterile insect technique and global trade. Entomologia Experimentalis et Applicata, 164, 204–225. [ Google Scholar ] Zepeda‐Cisneros, C.S. , Meza Hernández, J.S. , García‐Martínez, V. , Ibañez‐Palacios, J. , Zacharopoulou, A. and Franz, G. (2014) Development, genetic and cytogenetic analyses of genetic sexing strains of the Mexican fruit fly, Anastrepha ludens Loew (Diptera: Tephritidae). BMC Genetics, 15, S1. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] Articles from Insect Science are provided here courtesy of Wiley ACTIONS View on publisher site PDF (2.4 MB) Cite Collections Permalink PERMALINK Copy RESOURCES Similar articles Cited by other articles Links to NCBI Databases Cite Copy Download .nbib .nbib Format: AMA APA MLA NLM Add to Collections Create a new collection Add to an existing collection Name your collection * Choose a collection Unable to load your collection due to an error Please try again Add Cancel Follow NCBI NCBI on X (formerly known as Twitter) NCBI on Facebook NCBI on LinkedIn NCBI on GitHub NCBI RSS feed Connect with NLM NLM on X (formerly known as Twitter) NLM on Facebook NLM on YouTube National Library of Medicine 8600 Rockville Pike Bethesda, MD 20894 Web Policies FOIA HHS Vulnerability Disclosure Help Accessibility Careers NLM NIH HHS USA.gov Back to Top