You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Background
==========

*Anastrephaludens*has a major effect on México\'s economy because it attacks fruits as citrus and mangoes. In 2008 the exports of these two fruits only to the U.S. had a value of 121.8 million US dollars \[[@B1]\]. The Mexican federal government uses integrated pest management to control *A. ludens*through the Programa Nacional Moscas de la Fruta (SAGARPA-SENASICA). The sterile insect technique (SIT) is one of the main components of this program and involves mass-rearing and release of millions of sterile insects to reduce the birth rate in the target population \[[@B2]-[@B4]\]. This method has been successfully applied to control pests such as the screwworm fly, *Cochliomyia hominivorax*(Coquerel) and the Mediterranean fruit fly *Ceratitis capitata*(Wiedemann) \[[@B5],[@B6]\]. The main benefits of this method are that it is species-specific, does not generate biological or chemical pollution and is therefore environmentally friendly. The efficiency of the sterile insect technique increases if only males are released \[[@B7]\].

GSS have been constructed, in particular for *Ceratitis capitata*, and are used successfully in operational programs, demonstrating the following major advantages: 1. Economic savings in mass-rearing, irradiation, packing and release, 2. Increased efficiency in the field because sterile males compete better for wild females in the absence of sterile females 3. Increased fruit quality, avoiding damage caused by oviposition attempts by sterile females, and 4. In combination with a GSS, female-specific attractants in traps can be used thereby avoiding the re-capture of sterile males, reducing misidentification and simplifying and improving the accuracy of monitoring activities \[[@B8]-[@B10]\].

The construction of a GSS is based on two components: 1) A mutation that can be used as a selectable marker for sex separation; and 2) Y- autosome translocation linking the inheritance of this mutation to the sex, since the Y chromosome is responsible for the male sex \[[@B10],[@B11]\]. In a GSS, the wild-type allele of the selectable marker is physically linked to the Y chromosome through a Y-autosome translocation, while the females carry the mutant allele in homozygous condition. It should be pointed out that Y chromosome is carrying the Maleness factor, as it was shown in *C. capitata*and this probably holds for the whole Tephritidae family \[[@B12]\]. The main problems affecting the usefulness of GSSs based on Y-autosome translocations are:

1\) Genetic instability is due to pre-meiotic recombination in the parental male \[[@B13]\]. Although the recombination frequency is very low, accumulation and selection in favour of recombinants in the harsh environment of mass-rearing can lead eventually to the breakdown of the sexing system. This problem can be minimized by selecting a Y-autosome translocation where the autosomal breakpoint is close to the chromosomal location of the selectable marker \[[@B10],[@B11]\].

2\) Reduced product quality due to the survival of genetically unbalanced individuals resulting from adjacent-1 segregation during meiosis in the parental males \[[@B10]\]. This problem can be minimized by selecting a Y-autosome translocation where the adjacent-individuals die during early stages of development.

Genetic and cytogenetic analyses are necessary towards the selection of the most suitable GSS. The analysis of mitotic and polytene chromosomes provides detailed information on the structure of the chromosomal rearrangement, allowing the identification of the autosome(s) involved in the translocation and the mapping of chromosomes breakpoints. The evaluation of any new GSS is advisable before it should be used in mass-rearing \[[@B14]-[@B19]\].

In México, the sterile insect technique against the Mexican fruit fly *Anastrepha ludens*has been applied by releasing females and males because so far no GSS was available for that species. The development of the first genetic sexing system for this pest, based on the Y-autosome translocations and using the genetic marker *black pupae*(*bp*), is presented in this study.

Several GSS were constructed and tested, and one was selected because of its properties and potential to be used successfully in the Moscafrut mass-rearing facility.

Results
=======

Genetic analysis of the *black pupae*mutation
---------------------------------------------

The two reciprocal crosses, ♂*bp*^+^× ♀*bp*and ♂*bp*× ♀*bp^+^*, produced wild-type offspring.

Inbreeding the F~1~produced wild-type and mutant phenotypes at a ratio of 3:1 (Table [1](#T1){ref-type="table"}). From these results, we conclude that the *bp*mutation is recessive and autosomal. The correct classification of the colour of the pupae was confirmed based on the pigmentation of the adults. The abdomen, thorax and wings of *black pupae*adults are darker than the wild-type flies. In addition, the *black pupae*phenotype can be identified in the larval stage based on the black coloration of their anal lobes (Figure [1](#F1){ref-type="fig"}).

###### 

F~2~progeny of the cross between the wild-type and the *black pupae*strain

  ---------------------------------------------------------------------
  Parents     Cage            Wild-type   *black pupae*   X^2^   
  ----------- --------------- ----------- --------------- ------ ------
  Wild-type   *black pupae*   1           2,804           952    0.24

                              2           2,343           738    1.80

                              3           1,866           610    0.17

                                                                 

  *black*\    Wild-type       1           2,190           668    4.03
  *pupae*                                                        

                              2           2,266           759    0.13

                              3           1,761           572    0.29
  ---------------------------------------------------------------------

X^2^tables = 3.84, *P*= 0.05, df = 1

![**Larvae, pupae and adults from the **GSS***Tapachula -7*based on the *bp*mutation**. The males can be identified in the larval stage based on the brown coloration of their anal lobes compared to the black coloration of the females. a) *wild-type*males. b) *black pupae*females. Arrows point to the anal lobes.](1471-2156-15-S2-S1-1){#F1}

Development of genetic sexing strains
-------------------------------------

Ten genetic sexing strains were detected after screening 2,600 single male families, corresponding to a success rate of 0.38%. These strains were maintained in the laboratory for several generations. Females consistently emerged from black pupae, while males emerged from brown pupae (Figure [1](#F1){ref-type="fig"}).

Each strain had a specific average value of egg hatching. The strains T(Y:*bp*^+^)-7 T(Y:*bp*^+^)-3, T(Y:*bp*^+^)-1, T(Y:*bp*^+^)-10, T(Y:*bp*^+^)-6, T(Y:*bp*^+^)-2, T(Y:*bp*^+^)-8 and T(Y:*bp*^+^)-5 had the highest values, and their hatching ranged from 67.91% to 34.41%. The strains with the lowest values, T(Y:*bp*^+^)-4 and T(Y:*bp*^+^)-9 showed a hatch rate of 14.4% and 28.81%, respectively (Table [2](#T2){ref-type="table"}).

###### 

Evaluation during multiple generations of ten genetic sexing strains of *Anastrepha ludens*

  GSS   Number of generations evaluated   Pupae (no.)   Adults (no.)   Adults (%)   Egg hatch (%)   Recombinants (%)                             
  ----- --------------------------------- ------------- -------------- ------------ --------------- ------------------ -------- -------- ------- ------
  1     16                                17,343        15,060         15,580       82              34                 12,652   28,348   55.1    0.41
  2     15                                9,031         6,712          7,914        119             125                5,841    13,999   42.7    1.74
  3     8                                 6,545         6,438          5,283        188             96                 4,633    10,200   58.07   2.78
  4     9                                 2,074         1,973          1,889        1               0                  1,613    3,503    14.4    0.03
  5     6                                 4,641         4,172          3,817        0               0                  3,127    6,944    34.41   0
  6     6                                 7,362         4,742          5,893        0               0                  3,882    9,775    44.29   0
  7     7                                 10,765        9,843          9,592        0               0                  8,004    17,597   67.91   0
  8     5                                 2,738         2,614          2,358        0               32                 2,040    4,430    39.29   0.72
  9     5                                 1,367         2,059          1,171        1               4                  1,406    2,582    28.81   0.19
  10    6                                 4,825         4,634          4,510        3               17                 3,723    8,253    53.84   0.24

The ten strains analyzed also showed differences in the percentage of recombinant progeny. T(Y:*bp*^+^)-3, T(Y:*bp*^+^)-2 and T(Y:*bp*^+^)-8 had the highest values, with 2.78%, 1.74% and 0.72%, respectively. In contrast, the strains T(Y:*bp*^+^)-5, T(Y:*bp*^+^)-6 and T(Y:*bp*^+^)-7 did not show any recombinants at this level of rearing.

During this first evaluation, the strain T(Y:*bp*^+^)-7 showed a higher egg hatch as compared to the other GSS (Table [2](#T2){ref-type="table"}).

Survival test and genetic stability
-----------------------------------

The comparative analysis of the survival showed significant differences between the wild-type strain and the GSS T(Y:*bp*^+^)-7. The primary differences were observed at egg hatching (F = 135.46~2,\ 27~; *P*\<0.0001) and in larval survival (F = 32.37~2,\ 27~; *P*\<0.0001) while the differences in adult emergence were not significant (F = 1.69~2,\ 27~; *P*= 0.202) (Table [3](#T3){ref-type="table"}). A comparison of the egg to adult survival between the wild-type and the translocation strain shows that T(Y:*bp*^+^)-7 is roughly 50% sterile (68.5% as compared to 39.3%, Table [3](#T3){ref-type="table"}). Based on the results obtained with the medfly \[[@B20]\] this shows that T(Y:*bp*^+^)-7 carries a simple, reciprocal Y-autosome translocation, i.e. only one autosome is linked to the Y chromosome.

###### 

Survival of the wild-type and the *black pupae*strain compared to the genetic sexing strain T(Y:*bp*^+^)-7 (mean ± SD)

  ---------------------------------------------------------------------------------------------------------------------------------------
  Strain           Egg hatched\    Larval survival 3^rd^instar\   Larval to pupal survival\   Adult emergence\   Egg to adult survival\
                   (%)             (%)                            (%)                         (%)                (%)
  ---------------- --------------- ------------------------------ --------------------------- ------------------ ------------------------
  Wild type        94.60 ± 3.20a   73.40 ± 11.73a                 98.48 ± 1.12a               94.72 ± 4.30a      68.50 ± 12.00a

  *bp/bp*          91.80 ± 4.28a   76.20 ± 7.34a                  93.94 ± 5.74b               91.23 ± 4.44a      65.30 ± 8.12a

  T(Y:*bp*^+^)-7   63.70 ± 6.00b   43.90 ± 5.91b                  98.66 ± 2.92a               91.02 ± 6.16a      39.30 ± 5.25b
  ---------------------------------------------------------------------------------------------------------------------------------------

The same letter after the mean values indicates that the differences between these lines are not significant.

In the first experiment to determine the genetic stability of T(Y:*bp*^+^)-7, the strain was reared with 600 to 2500 parental pairs per generation and no recombinants were observed (Table [4](#T4){ref-type="table"}). However, in the second experiment where the population was increased averages of 0.025% recombinant insects were found per generation (Table [5](#T5){ref-type="table"}). A third, larger scale experiment (15,000 parental pupae per generation) was set up but this time recombinants were not removed. The highest level of recombinants reached 1.5% in the eighth generation (Table [6](#T6){ref-type="table"}).

###### 

Progeny of the strain T(Y:*bp*^+^)-7 during multiple generations (F) with different numbers of breeders

  F    No. of pairs   No. of pupae   Total pupae   Adults *bp*   Adults *bp^+^*   Total insects                                    
  ---- -------------- -------------- ------------- ------------- ---------------- --------------- ----- ---- ------- --- ---- ---- -------
  14   2,500          2,481          2,913         5,394         1,625            0               \_    0    2,219   0   \_   \_   3,844
  15   1,500          3,000          3,000         6,000         2,476            0               74    21   2,750   0   19   13   5,226
  16   1,500          2,600          2,800         5,400         2,059            0               85    19   2,492   0   25   10   4,551
  17   600            690            700           1,390         610              0               \_    \_   605     0   \_   \_   1,215
  18   1,500          2,310          2,665         4,975         1,894            0               102   37   2,392   0   59   28   4,286
  19   1,500          2,607          2,659         5,266         2,222            0               76    24   2,415   0   15   13   4,637
  20   1,500          3,000          3,000         6,000         2,507            0               106   34   2,809   0   29   12   5,316

###### 

Progeny of the strain T(Y:*bp*^+^)-7 during eight generations of rearing with 3000 females and 2500 males per cage (recombinants were removed each generation)

  -------------------------------------------------------------------------------------------------
  Adults *bp*   Adults *bp^+^*   Total insects   Recombinants\                               
                                                 (%)                                         
  ------------- ---------------- --------------- --------------- ------ --- ---- ---- ------ ------
  3549          0                123             19              2698   0   50   15   6281   0.00

  3455          0                130             6               2802   1   39   7    6271   0.02

  3916          0                64              14              2698   0   14   7    6635   0.00

  3842          1                68              15              2755   0   24   6    6619   0.02

  3365          3                89              24              2633   0   32   12   6037   0.05

  3517          2                49              12              2600   1   14   7    6139   0.05

  3487          1                96              43              2564   1   43   14   6110   0.03

  3227          1                53              49              2457   1   33   34   5769   0.03
  -------------------------------------------------------------------------------------------------

###### 

Progeny of the strain T(Y:*bp*^+^)-7 during ten generations without removing recombinants

  F       No. of pupae   Total pupae   Adults *bp*   Adults *bp^+^*   Total insects   Recombinants (%)                                       
  ------- -------------- ------------- ------------- ---------------- --------------- ------------------ ----- ------ ---- ----- ---- ------ ------
  P       935            1072          2007          709              0               20                 3     1009   0    9     2    1718   0.00
  F~1~    1058           1450          2508          1045             0               14                 16    1311   0    15    5    2356   0.00
  F~2~    1299           1348          2647          1142             0               16                 9     1262   0    16    6    2451   0.00
  F~3~    1185           1499          2684          1026             0               12                 4     1410   1    12    8    2473   0.04
  F~4~    1178           1198          2376          1051             0               6                  3     1159   0    6     5    2230   0.00
  F~5~    1135           1304          2439          856              0               12                 8     1009   0    15    12   1912   0.00
  F~6~    1294           1405          2699          919              0               146                49    1023   0    60    19   2216   0.00
  F~7~    1134           1408          2542          849              0               33                 7     1103   0    22    9    2023   0.00
  F~8~    8000           7000          15000         5016             16              260                116   4313   14   102   48   9885   0.30
  F~8~    834            968           1802          606              5               17                 41    808    17   6     12   1512   1.50
  F~9~    831            1188          2019          641              1               37                 17    1027   9    7     4    1743   0.57
  F~10~   938            1096          2034          713              0               12                 3     986    13   6     3    1699   0.76

Cytogenetic analysis
--------------------

**Mitotic chromosomes**. *Anastrepha ludens*has six pairs of acrocentric chromosomes, five autosomes and a pair of sex chromosomes (Figure [2a](#F2){ref-type="fig"}) \[[@B21]\].The male is heterogametic (XY) \[[@B21]\]. The autosomes display the characteristic somatic pairing observed in Diptera and this is very useful for the identification of homologous chromosomes. The analysis of at least 80 cells from the GSS T(Y:*bp*^+^)-7 indicates that the translocation involves the largest autosome (chromosome 2) and the Y chromosome. As it is evident from Figures [2b,c](#F2){ref-type="fig"} a small distal part of this autosome is joined to the Y chromosome, while a tiny part of the Y chromosome is connected to the autosomal breakpoint. The resulting 2-Y translocation chromosome is slightly shorter (10.58 ± 0.99%) than the wild-type chromosome 2 (11.93 ± 0.8%). The reciprocal Y-2 translocation fragment in the strain T(Y:*bp*^+^)-7 is longer than the wild-type Y chromosome (2.75 ± 0.37% versus 1.71 ± 0.3%). A graphic representation of the Y-autosome translocation is shown in Figure [2d](#F2){ref-type="fig"}.

![**a) *Anastrepha ludens*mitotic metaphase chromosomes from brain ganglia of third instar male larva of the wild-type strain**. b-c**)**Mitotic metaphases of the GSS *Tapachula-7*. d**)**Graphic representation of the Y-autosome translocation observed in the *Tapachula-7*strain and its comparison with the wild-type. e-f) Mitotic metaphases of the second GSS, *Tapachula -1*. X, Y: represent the sex chromosomes, 2: represents the intact free autosome, Y-2: represents the translocation fragment with the Y centromere, 2-Y: represents the reciprocal translocation fragment carrying the autosomal centromere. Centromeres are indicated by circles in 2d. *Bar*= 10µm](1471-2156-15-S2-S1-2){#F2}

The analysis of the mitotic chromosomes in another GSS strain, T(Y:*bp*^+^)-1, showed that the same autosome, chromosome 2, is involved in the translocation. However, the autosomal breakpoint seems to be at a different position because the 2-Y fragment was here 9.08 ± 0.62% and the Y-2 fragment 3.77 ± 0.31% long (Figure [2e-f](#F2){ref-type="fig"}).

**Polytene chromosomes**. Polytene chromosomes were analyzed in male larvae from the GSS T(Y:*bp*^+^)-7. The analysis was focused only on males where the reciprocal translocation can be observed. Although the Y chromosome cannot be identified in polytene nuclei using standard staining, we succeeded to identify the autosome involved in the translocation and map the autosomal breakpoint. *Anastrepha ludens*shows a characteristic ectopic pairing between the telomeres of the autosomes \[[@B21]\], a phenomenon that also was observed in other Tephritidae species as *Bactrocera oleae*\[[@B22]\] and *B. cucurbitae*\[[@B23]\]. One of the most frequent ectopic pairing has been observed between the polytene elements III and V (Figure [3f](#F3){ref-type="fig"}). Polytene chromosomes from the strain T(Y:*bp*^+^)-7 are shown in Figure [3a-e](#F3){ref-type="fig"}. The ectopic pairing of the two telomeres, chromosome III and V are shown, but interestingly only one homolog of the polytene element III is involved in this. In all cases, a part of a single chromosome III is connected with its telomere to the telomere of chromosome V. This region should represent the distal part of the chromosome element involved in the Y-autosome translocation. In this ectopic pairing either the telomere of the intact chromosome III is taking part (Figure [3a](#F3){ref-type="fig"}); or the fragment of the chromosome that is connected to the Y chromosome (Y-A) (Figure [3b-e](#F3){ref-type="fig"}) is pairing. Based on these observations as well as on the mitotic karyotype of this strain, we can conclude that polytene chromosome III corresponds to the longest chromosome of the mitotic karyotype, i.e. chromosome 2. Taking into account both the mitotic karyotype and polytene chromosomes analyses of this GSS, we can propose the structure of the T(Y:*bp^+^*)-7. According to the chromosome maps of *A. ludens*\[[@B21]\] the chromosome III has a breakpoint at the beginning of the region 25 (Figure [4](#F4){ref-type="fig"}). This breakpoint produces two autosomal fragments of different size: The short fragment includes the distal part of the chromosome from the telomere to 25 chromosomal region (24.67 µm) while the second fragment consisting of the rest of the chromosome element III. The short fragment is joined to that part of the Y chromosome that carries the centromere Y-2 (Figure [2d](#F2){ref-type="fig"}). The second autosomal fragment, with the autosomal centromere, is linked to remaining region of the Y chromosome resulting in the 2-Ychromosome (Figure [2d](#F2){ref-type="fig"}) that is slightly shorter than the wild-type chromosome element III. Each of these new chromosomes, 2-Y and Y-2 has its centromere and telomere sufficient for their stability.

![**Polytene chromosomes from the strain T(Y:*bp*^+^)-7**. Thick arrows show the ectopic paring between the telomeres of the V and III chromosomes. a) Thin arrow shows the autosome breakpoint; in this case the intact chromosome III is taken part in the ectopic pairing. b-e) The autosome fragment of the Y-A chromosome is taken part in the ectopic pairing. f) Normal ectopic pairing between chromosomes V and III from the wild-type strain. *Bar*= 10µm](1471-2156-15-S2-S1-3){#F3}

![**a) The short translocation fragment representing the Y-2 chromosome of mitotic metaphases or Y-III of polytene chromosomes**. b) Reference map of chromosome III showing the region involved in translocation; arrow in the map shows the position of the breakpoint. *Bar*= 10µm](1471-2156-15-S2-S1-4){#F4}

Discussion
==========

We have developed a GSS for *Anastrepha ludens*using the *black pupae*mutation as a selectable marker. In this strain, males emerge from wild-type (brown) pupae, whereas females emerge from mutant (black) pupae. This particular mutation has additional benefits because it is also expressed in the larval and the adult stage. This characteristic confers various benefits to this system, including greater reliability, avoiding the difficulties in the clear discrimination of the colours as it was reported by Rössler \[[@B24]\] for the genetic sexing system based on the marker *dark pupae*in *Ceratitis capitata*. Undoubtedly, the genetic marker *white pupae*found in other fruit flies provide better separation due to the greater contrast with the wild-type pupae \[[@B25]-[@B30]\].

However, despite significant efforts, this marker has not been found yet in *Anastrepha ludens*, neither in field populations nor in inbred laboratory colonies.

During this study, different doses of irradiation were used: 25 Gy, 30 Gy and 40 Gy. The first dose was too low, and no translocations were obtained. The highest dose induced such a high sterility that it was impossible to breed the insects. The optimal dose was 30 Gy, and the success rate was 0.38% (10/2600). The doses used to induce translocations were lower than those used in *Ceratitiscapitata*. Franz et al., (1994) \[[@B31]\] reported a success rate of 7.1% at 50 Gy, and Kerremans and Franz, (1995) \[[@B17]\] reported a success rate of 4.3% at 40 Gy. These differences between *C. capitata*and *A. ludens*indicate that it is necessary to determine the optimal dose for each species individually. The optimal dose can be influenced by specific biological characteristics, such as the size of the Y chromosome. The Y chromosome of *C. capitata*is significantly longer than that of *A. ludens*and this might be responsible for a greater probability for inducing Y-autosome translocation compared to *A. ludens*.

Genetic stability is one of the most important aspects in the evaluation of a GSS and its usefulness for a practical application. Genetic stability depends directly on the structure of the translocation, i.e. it depends on the distance between the autosomal breakpoint of the translocation and the location of the genetic marker \[[@B31]\]. This distance is directly proportional to the frequency of recombination in this chromosomal region in the heterozygotic males of a sexing strain \[[@B13]\]. The recombinant flies show a reverse phenotype as compared to the normal, non-recombinant flies, i.e. recombination in the parental males produces black pupae males and brown pupae females. The percentage of recombinant flies during various generations was high in the strains T(Y:*bp*^+^)-2, T(Y:*bp*^+^)-3 and T(Y:*bp*^+^)-8. These strains were considered unstable. On the contrary, the strain T(Y:*bp*^+^)-7 showed no recombinants when reared at a smaller scale. However, the strain showed 0.025% of recombinants per generation when the scale of rearing was increased for several generations. Monitoring a larger number of insects over a longer period of time will provide additional results to predict the behaviour of this strain under mass-rearing conditions.

In the evaluation of a GSS, it is important to consider the productivity and the product quality, both of which are tightly linked to the structure of the translocation. The segregation behaviour of the translocation during male meiosis will determine the number of genetically balanced or unbalanced individuals in the next generation. During male meiosis simple Y-autosome translocations can segregate in two different ways if they follow the rule that homologous centromeres segregate: alternate or adjacent-1 segregation. In the former the two translocation chromosomes stay together and segregate from the X chromosome. In the latter the A-Y chromosome segregates together with the X chromosome while the Y-A chromosome segregates with the non-translocated autosomal homologue. Figure [5](#F5){ref-type="fig"} shows, that after fertilization only alternate segregation results in genetically balanced offspring while adjacent-1 segregation results in offspring where the chromosomal segment between the translocation breakpoint and the tip of respective chromosome arm is either present in three copies or segregation only in a haploid condition. At least in the medfly such long deletions lead to early lethality which is manifested as reduced egg hatch. The triplication type adjacent-1 offspring can survive, depending on the length of the triplication, even to the adult stage, although at severely reduced numbers \[[@B12]\]. Only if the autosome breakpoint is very close to the tip, i.e. the triplication is very short, such flies mate and produce offspring (Franz, personal communication). Both forms of segregation are completely legitimate and that explains why they occur at equal frequency in most translocation strains, at least in medfly. Consequently, only half of the sperm produced by males carrying a Y-autosome translocation lead to viable, genetically balanced offspring, i.e. such males are 50% sterile. In a third form of segregation, adjacent-2, homologous centromeres do not segregate, i.e. this is a form of non-disjunction and therefore significantly less likely to occur.

![Schematic representation of alternate and adjacent-1 segregation in males with a Y-autosome translocation](1471-2156-15-S2-S1-5){#F5}

The egg hatch in strain T(Y:*bp*^+^)-7 is reduced due to the lethality of the deletion type adjacent-1 offspring. In addition, also the triplication carrying adjacent-1 individuals should ideally die as early as possible to avoid wasting larval diet and to avoid confusions in the quality control. In the T(Y:*bp*^+^)-7 strain, this seems to be the case as larval survival is the only parameter besides egg hatch that differs significantly from the wild-type level.

The cytogenetic studies performed using mitotic chromosomes showed that T(Y:*bp*^+^)-7 carries a simple reciprocal translocation between the longest autosome, chromosome 2, and the Y chromosome. Analysis of polytene chromosomes permitted us to identify the polytene element involved in the translocation. Based on the specific ectopic pairing observed between the telomeres of autosomes in the *A. ludens*polytene nuclei \[[@B21]\], we show that chromosome 2 of the mitotic karyotype corresponds to polytene element III (Figure [6](#F6){ref-type="fig"}). In addition, this analysis permitted us to map the autosomal breakpoint on chromosome III despite the fact that the Y chromosome remains underreplicated in polytene nuclei and cannot be identified after standard staining. The autosomal breakpoint is close to the beginning of region 25 of chromosome III according to the polytene maps of the *A. ludens*(Figure [4](#F4){ref-type="fig"}). It should be emphasized that both translocation chromosomes possess centromeres and telomeres, required for stability during cell divisions.

![**Schematic representation of the correlation between mitotic chromosome 2 and polytene chromosome III in *wild-type*and *Tapachula-7*strain showing the induced translocation in mitotic (2-Y) (Y-2) and the respective polytene chromosome (III-Y) (Y-III)**. Y represents sex chromosome.](1471-2156-15-S2-S1-6){#F6}

Ideally, a genetic sexing system allows the elimination of females during the early stages of development to decrease the cost during mass-rearing effectively and to increase the capacity of the production facility. However, the method of sex separation we report here is based on the coloration of the pupae and allows sexing only at the pupal stage. This is done mechanically using sorting machines available on the market. These machines can be adapted for this specific use by modifying their sorting characteristics so that brown and black pupae can be separated with the required speed and accuracy. In 1969, Whitten \[[@B32]\] suggested this type of mechanized separation for GSS based on the colour of pupae to obtain sufficient quantities to meet the demand of an area-wide control program. The advantage of this method of separation is that it is non-destructive, allowing to feed the females back into the rearing colony and, thereby, increasing production capacity if required \[[@B33]\]. Using such a seed sorting machine, Robinson and Riva \[[@B34]\] separated white and brown pupae of a Mediterranean fruit fly GSS with a success rate of 100%.

Conclusions
===========

We have isolated the first GSS in *A. ludens*, which was given the common name *Tapachula-7*. Its characteristics, as far as we have determined them in the research described here, indicate that this strain will be stable and productive enough for a practical application in the mass-rearing for field operations. However, further testing under mass-rearing conditions is required to come to a final conclusion. In addition, the results of the current study show that mitotic and polytene chromosomes of this species are suitable for cytogenetic studies and could support the development of improved control methods in this pest.

Materials and methods
=====================

Biological material
-------------------

The colony of *Anastrepha ludens*used in this study was founded using flies collected in the wild in the state of Chiapas, México \[[@B35]\]. The insects were then kept in the laboratory for several generations with photoperiods of 12:12, a temperature of 26°C and a relative humidity of 70-80%.

*black pupae*strain
-------------------

Pupae with black colour, distinct from the brown-coloured wild-type pupae, were isolated from the mass-rearing facility Moscafrut located in Metapa de Domínguez Chiapas, México after screening approximately 7, 700 000.00 pupae. Male and female adults derived from *black pupae*had noticeably darker cuticles and wing lines than the wild-type individuals. Larval anal lobes were black and easy to be differentiated from the wild-type \[[@B36]\].

Rearing the flies
-----------------

Adults are maintained in laboratory conditions at 26°C and a relative humidity of 60-70% with *ad libitum*water and a mixture of sugar and hydrolyzed proteins (yeast hydrolysate enzymatic MP Biomedicals, LLC France). Eggs are collected and incubated in Petri dishes with saturated humidity for 4 days at 26°C in a bioclimatic chamber Binder KBF720. The larvae are fed with diet of powdered corncob (19% powdered corncob, 9.2% sugar, 5.3% corn flour, 7.0% yeast, 0.44% citric acid, 0.4% sodium benzoate, 0.2% nipagin and 0.1% guar gum) and are maintained under controlled temperature and humidity. Pupation is carried out over 14 days on Vermiculite Strong Lite^®^substrate.

Genetic analysis
----------------

The inheritance of the *black pupae*mutation was determined by crossing wild-type males to *black pupae*females and *black pupae*males to wild-type females (♂wt to ♀*bp*, ♂*bp*to ♀wt). Three cages were set up for each cross. Eggs were collected from each cage, and the breeding process was continued until the emergence of the F~1~generation progeny. Five females and 5 males of the F~1~generation were then inbred to obtain the F~2~generation. F~1~and F~2~pupae were carefully checked and separated by colour, and the adults were observed under a Zeiss stereomicroscope to confirm their phenotype.

Development of genetic sexing strain
------------------------------------

Wild-type pupae two days before emergence were irradiated with 30 Gray (Gy) using a cobalt-60 Gammacell 220 source. The irradiated males were then placed in 4 litre cages and crossed with virgin females homozygous for *black pupae*(*bp/bp*).

The F~1~males of that cross were singly backcrossed to mutant females. If the F~1~males carry a translocation between the Y and the autosome bearing the selectable marker \"*bp*\", all the males of a particular family will be wild-type, and all the females will be mutant (Figure [7](#F7){ref-type="fig"}). Where such a family was detected in the screen, the progeny was further propagated, and their true-breeding nature was ascertained and maintained in the laboratory for several generations. In each generation, the percentage of the hatched eggs, the number of pupae and their colour as well as the number of adults were recorded. When recombinants, i.e. females emerging from brown and males from black pupae, were detected they were counted and removed. The pupal colour separation was performed manually, and the correct classification was corroborated in adults using the dark coloration of the *black pupae*strain for differentiation.

![**Crossing scheme for the isolation of genetic sexing strains in *A. ludens*.** X and Y: represent the sex chromosomes. *bp^+^*: represents the autosome harboring the wild type allele for the brown pupae color. *bp*: represents the autosome harboring the *blackpupae*(*bp*) mutation. Y-2 and 2-Y: represent the two new chromosomes following the translocation event.](1471-2156-15-S2-S1-7){#F7}

Survival test and genetic stability
-----------------------------------

The following three types of crosses were set up: a) flies from the *black pupae*strain (100♀ and 100♂) b) wild-type (50♀ and 50♂) and c) the GSS *Tapachula-7*(100♀ and 100♂). Each one was placed separately in cages of 30x30x30 cm. When the insects reached sexual maturity, 1000 eggs were taken in batches of 100. The eggs were incubated for 4 days at 26 ± 1°C in a bioclimatic KBF720 Binder chamber, and the number of fertile eggs was quantified after 7 days. The larval development of 100 eggs was conducted in a Petri dish with the specified larval diet. The number of third-instar larvae was quantified after 10 days. The number of mature pupae and emerged adults was also recorded.

The T(Y:*bp*^+^)-7 was reared for some generations increasing the number of parents. Three experiments were carried out: the first was using a population in the cage of 600-2500 adults, evaluated during seven generations and in each generation the recombinants were removed and the pupae yielded were recorded. In the second experiment the population used was 3000 females × 2500 males and recombinants were monitored during eight generations, removing the recombinants in each generation. In the third experiment we monitored ten generations of rearing with 15,000 parental pupae (mixed black and brown) per generation in large cages (the recombinants were not removed during this experiment). To determine the number of recombinants in each generation, a sample of 100 ml of pupae was analyzed in each generation.

Cytogenetic analysis
--------------------

Mitotic and polytene chromosomes were prepared from the genetic sexing strain T(Y:*bp*^+^)-7. Metaphase chromosomes were prepared from the brain ganglia of third-instar larvae \[[@B20]\] while for the staining the application of the C- banding technique was used \[[@B37]\].

The slides were immersed in hydrochloric acid 0.2 N for 90 s, washed with distilled water and immersed in 5% barium hydroxide at 40°C for 1 min. The slides were then washed in distilled water with a few drops of acetic acid and placed in 2xSSC at 60°C for 30 min. Finally, the slides were washed and stained using 25% Wright stain in Sörensen buffer for 1 min and 12% Giemsa stain for 1 hour. The samples were observed using a Zeiss Axioskop 40 microscope with 100x magnification objective, and images were acquired using the software Axiovision 4.8.2 by Carl Zeiss de México S.A. de C.V. The length of the chromosomes in 35 metaphases from the wild-type samples and 15 from the genetic sexing strain were measured with UTHSCSA Image tool 3.0 (web site <http://compdent.uthscsa.edu/dig/itdesc.html>). Only well metaphase spreads with 12 chromosomes were used to measure the total length of each chromosome. The relative chromosomal length was estimated as the percentage of the length of each chromosome relative to the total length of the total diploid complement (%RL). Third instar male larvae were used for the salivary gland polytene chromosomes using the method described for *C. capitata*\[[@B18],[@B38]\] and previously used for *A. ludens*by García-Martínez et al. \[[@B21]\]. The male larvae were identified based on the brown coloration of the anal lobes compared to the black coloration of the female larvae.

Data analysis
-------------

The overall fitness was estimated by multiplying the transformation frequency between the stages (proportion of eggs hatched, larvae survival, pupae survival and pupae to adult survival). The data were transformed by arcsine √x and analyzed by ANOVA. Medians were compared using Tukey\'s test with α = 0.5. Both of these analyses were performed using the software JMP version 5.0 (web site <http://www.jmp.com>).

Competing interests
===================

The authors declare that they have no competing interests.

Authors\' contributions
=======================

CSZC directed the study, designed the protocol, selected the data to present, integrated and composed the manuscript and collaborated with VGM to implement novel technical modifications for the study of mitotic chromosomes.

JSMH designed protocols for the construction and testing of the genetic sexing strains, integrated and analyzed the data, performed the statistical analysis and help to integrate the initial draft of the manuscript.

VGM conducted the laboratory cytogenetic methods, including slide preparation and imaging, discussed the results of the cytogenetic analysis and revised the manuscript.

JIP coordinated the laboratory work for establishing protocols and evaluating the genetic sexing strains.

AZ participated in the interpretation of the cytogenetic analysis data and reviewing the manuscript.

GF designed the protocols for constructing the genetic sexing strains and reviewing the manuscript.

Acknowledgements
================

The authors would like to thank the International Atomic Energy Agency for the financial and technical support provided though the National Project MEX/5/027 and the Coordinated Research Project No. 15682.

This article has been published as part of *BMC Genetics*Volume 15 Supplement 2, 2014: Development and evaluation of improved strains of insect pests for SIT. The full contents of the supplement are available online at <http://www.biomedcentral.com/bmcgenet/supplements/15/S2>.

Publication of this supplement was funded by the International Atomic Energy Agency. The peer review process for articles published in this supplement was overseen by the Supplement Editors in accordance with BioMed Central\'s peer review guidelines for supplements. The Supplement Editors declare that they have no competing interests.