Sterile Insect Technique (SIT) applications against major insect pests and disease vectors rely on the cost-effective production of high-quality sterile males. This largely depends on the optimal management of target pest colonies by maximizing the benefits provided by a genetically rich and pathogen-free mother colony, the presence of symbiotic microorganisms, and efficient domestication, mass-rearing, irradiation, and release processes. At the same time microbial (bacteria, fungi, microsporidia, and viruses) pathogen outbreaks should be minimized or eliminated, and the use of hazardous chemicals restricted. The optimization of the colony management strategies for different SIT target insects will ensure a standardized high-quality mass-rearing process and the cost-effective production of sterile males with enhanced field performance and male mating competitiveness. The aims of the Coordinated Research Project (CRP) were to develop best practices for insect colony management for the cost-effective production of high-quality sterile males for SIT applications against major insect pests and disease vectors through a multidisciplinary approach involving entomologists, geneticists, ecologists, microbiologists, pathologists, virologists, and mass-rearing experts.

Stress may be viewed as the disturbance of homeostasis of an organism. Stress may arise from the external or internal environment of living organisms and plays a significant role in the fight or flight responses of animals. An organism's potential to resist stress is determined by its ability to mount up an effective response against the stress factors. Therefore, stress-induced biomolecules are useful indicators of a well-functioning immune system. While the interactions between stress and immunity are well-studied in vertebrate and plant systems, they are insufficiently documented among invertebrates, including dipteran insects that are mass-reared for sterile insect technique (SIT) applications. Generally, mass-reared insects may experience a variety of stress factors, which may affect various biological traits, including fecundity, weight of the progeny, adult emergence rates, flight propensity, mating ability, and their competitiveness with wild conspecifics. Many of these biological traits determine the costs and success of SIT programs. It is imperative to understand how stress impacts the quality of the reared insects and their biological traits, as well as the insect's defense responses to stress factors, to maintain robust and healthy colonies for successful release programs in SIT. Here, we review and discuss the sources and responses to biotic and abiotic stress in general in insects, while prioritizing literature on dipteran insects for SIT programs. We also coalesce genes and pathways that are modulated during stress and may be used as indicators to diagnose stress with the final aim to improve insect health in mass-rearing colonies.

The sterile insect technique (SIT) is a pest control method that has been successful in controlling various species of fruit flies of economic importance worldwide. However, SIT procedures can affect the performance of mass-reared sterile fruit flies, compromising the SIT effectiveness. This review aims to identify and analyze the adverse effects associated with each step of the SIT. Special emphasis is placed on identifying critical points in the SIT process that compromise the quality and highlighting the stages that require greater optimization to improve the effectiveness of the technique. We reviewed the main scientific findings that document the impact of domestication, mass-rearing, irradiation, handling, and release on the biology, ecology, behavior, and genetic makeup of sterile males, and how they affect the performance of sterile males under natural conditions, and the effectiveness of the SIT. Although all stages of the Sterile Insect Technique (SIT) impose some degree of compromise on insect performance, domestication and mass-rearing have the most profoundly negative impact on the field performance of sterile fruit flies. As the initial and most influential stages, they largely determine the overall quality and competitiveness of released insects, exerting a stronger impact than any subsequent SIT component.

For almost a decade, natural populations of the South American fruit fly have been targeted for control through Sterile Insect Technique projects. To ensure a sustainable supply of competitive sterile flies for this approach, it is essential to understand the effects of domestication when strains of this pest are initially brought into the laboratory to establish colonies as well as the changes occurring after multiple generations of adaptation to conditions used for mass rearing. Using one colony established from a wild population of the Brazil-1 morphotype (WIL) and two from laboratory colonies in Brazil known as the Piracicaba (PL) and Vacaria (VL) strains, this study evaluated genetic diversity in samples from 10 generations after domestication and maintenance under semimass rearing conditions. Another aim of this study was to analyze changes in the genetic makeup of the colonies of the two laboratory strains after refreshment. Eight microsatellite markers were used for the genetic analyses. Results indicated a moderate but significant amount of genetic differentiation between the WIL population and the two laboratory strains. Results also showed that levels of genetic diversity in both the VL and PL strains were maintained at similar levels over a period of more than two years of rearing. Additionally, results suggest that successful creation of admixture via refreshment is more likely to be beneficial in relatively short-term domesticated colonies, and that performing refreshment approximately every six to eight generations could be beneficial to maintain the genetic diversity of A. fraterculus colonies under laboratory mass rearing conditions.

Tsetse flies are the sole cyclic vectors of African trypanosomes, which cause human and animal African trypanosomiases in Africa. Tsetse fly control remains a promising option for disease management. The sterile insect technique (SIT) stands as an environmentally friendly tool to control tsetse populations. SIT requires the mass-rearing of competent sterile males to mate with wild females. However, long-term colonization might affect the genetic structure of the reared flies. This study investigated the genetic structure of four Glossina palpalis gambiensis colonies of different ages: two originating from Senegal (SEN and ICIRSEN) and two from Burkina Faso (CIR and IBD). Samples from these colonies were genotyped at ten microsatellite loci, followed by downstream population genetic analyses. The results show that the two colonies from Burkina Faso collected from close sites (∼20 km apart) over 45-year interval retained the same genetic background (F_{ST_CIR∼IBD} ≈ 0, P-value = 0.47). These flies were however, genetically different from those from the Senegal colonies (F_{ST_CIR∼SEN} ≈ 0.047; F_{ST_IBD∼SEN} ≈ 0.058, P-value = 10⁻⁴). Moreover, no significant difference was detected in the gene diversity of the CIR and IBD colonies, with H_S values of 0.650 and 0.665, respectively. The inbreeding coefficient showed that all four colonies where under Hardy–Weinberg equilibrium, with F_IS values of 0.026, 0.012, −0.064, and 0.001, for CIR, IBD, ICIRSEN, and SEN, respectively. Furthermore, no sign of a recent bottleneck was identified in tsetse samples from any of the four colonies. The results suggest that long-term mass-rearing of tsetse flies has no significant impact on their genetic background and diversity.

Tsetse (Glossina spp.) are vectors of African trypanosomes that cause devastating human and animal African trypanosomiases. While much of the research to better understand tsetse genetics and physiology relies on colony-reared flies, these flies may not represent the genetic diversity found in natural wild populations due to their long-term captivity. To enhance the translation of colony research into field applications, we utilized Nanopore sequencing to assemble genomes for a wild-caught female Glossina fuscipes fuscipes (Gff) from northwestern Uganda and for a female Gff from a laboratory line originally sourced from the Central African Republic in 1986. The new assemblies, from the wild-caught Gff (405.98 Mb, N50: 56.86 Mb) and the laboratory-derived Gff (398.22 Mb, N50: 47.811 Mb), demonstrate near-chromosomal level contiguity, high BUSCO scores (> 99.5%), high QV scores (> 37), and over 12 345 genes. Alignments between both new genomes reveal conserved synteny with only minor structural variants in the X, 1L, 1R, 2L, and 2R tsetse chromosomes. While most orthologs (10 730) were shared between both new genomes, we identified 381 unique orthologs and a small number of highly diverged shared single-copy homologs (3.84%). These gene-set differences could represent population-level variation due to the distinct geographic origin of these flies or adaptation to colony conditions. Our new high-quality genomes, with improvements in contiguity and completeness compared to the current NCBI RefSeq Gff genome, lay the foundation for advanced tsetse research, enabling robust lab-to-field translational applications to deepen our understanding of vector biology and disease transmission dynamics.

The sterile insect technique (SIT) for controlling the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), requires the mass-rearing of males in bio-facilities. This study summarizes the implementation of a Pre-Filter system in the mass-rearing process of C. capitata at the Moscamed program's El Pino facility in Guatemala. The Pre-Filter selection process as presented here was aimed to increase the longevity of the VIENNA 8 D53- strain males released for pest control. Over a 9-year period (2015–2024), the longevity of these males was systemically monitored, and the impact of the Pre-Filter system on mortality, half-life, and survival rate were analyzed. Results indicated a significant reduction in mortality at 48 h, declining from 16.47% in 2017 to 2.56% in 2024. Concurrently, the half-life of males increased from 79.55 h in 2018 to 85.97 h in 2024. The Pre-Filter system shifted the frequency distribution of half-lives toward longer durations, with a peak observed between 76.14 and 86.14 h, whereas populations without the Pre-Filter exhibited shorter half-lives, peaking between 56.14 and 66.14 h. Comparative analyses with historical data (2005–2024) demonstrated that the Pre-Filter system significantly improved the male longevity, compared to pre-implementation periods during which mortality rates were higher and more variable. Additionally, the results revealed a gradual reduction in longevity variability within the population, indicating a more homogeneous longevity among the mass-reared males.

In cases where a pest that is target of a Sterile Insect Technique (SIT) campaign is a member of a cryptic species complex, it is necessary to know in advance whether the sterile mass-reared males are sexually compatible with the wild females, otherwise the releases would result in failure to induce sterility in the target population. The South American fruit fly, commonly known as Anastrepha fraterculus, represents such a complex of cryptic species with at least 8 different morphotypes. From northern Argentina to southeastern areas of Brazil, the “Brazil-1” morphotype predominates and laboratory colonies have been established for its control through the use of SIT. Our goal was to assess the mating compatibility between different populations, including two wild ones from southern Brazil (Vacaria-WV; and Pelotas-WP) and laboratory strains derived from this morphotype. These included two bisexual laboratory strains (Piracicaba-PIRA; and Vacaria-VAC), and a recently developed genetic sexing strain, the GSS-89. Field cage tests with fertile flies demonstrated that PIRA flies present partial sexual incompatibility with all other strains, and therefore cannot be recommended for SIT field release. Also, males of the VAC strain, both fertile and sterile, mated randomly with WV and WP flies. No evidence of sexual isolation was found between the flies of the GSS and WV flies, but a certain level of incompatibility was shown between the fertile or sterile GSS males and WP females. Recommendations on the use of those strains through the SIT against the two southern Brazilian populations were made.

Anastrepha fraterculus is a significant fruit fly pest in Argentina and other South American countries. Previous studies showed the key role of gut bacteria in the protection and nutrient assimilation of fruit flies, particularly the importance of the biological fixation of nitrogen (diazotrophy). The presence of diazotrophic bacteria in A. fraterculus sp. 1 has been demonstrated through molecular, culture-independent methods. This study is aimed to characterize the composition and diversity of culturable gut bacteria of A. fraterculus sp. 1 males from different origins, and explore their metabolic roles, focusing on diazotrophic bacteria. Three male groups were studied: wild-caught (WW), lab-reared from wild larvae (WL), and lab-colony raised (LL). Gut bacteria were collected and characterized via 16S rRNA gene sequencing, with potential diazotrophs screened using selective media (SIL and NFb). Phylogenetic analysis of 16S rRNA gene mapped potential diazotrophs across the bacterial collection, while biochemical profiling and ARDRA (Amplified rDNA Restriction Analysis) were used to quickly differentiate diazotrophic bacteria. PCR testing for the nifH gene, associated with nitrogen fixation, was also performed. Bacterial diversity was highest in WW, followed by WL, and lowest in LL. In LL and WL, Enterobacter was the most frequent genus, while Klebsiella dominated in WW. Among the 20 SIL+ isolates identified, 10 came from WW, 9 from WL, and 1 from LL. One of these isolates (Enterobacter sp.) was tested as a supplement to the adult diet, without showing a beneficial effect on males pheromone calling behavior. Three isolates were also NFb+; two had the nifH gene. ARDRA was effective for rapid diazotroph discrimination. These findings highlight the potential of gut symbiotic bacteria in eco-friendly pest management strategies like the sterile insect technique (SIT). By using diazotrophic bacteria, protein requirements in artificial diets could be reduced, cutting costs and improving the affordability of SIT programs.

The Mexican fruit fly, Anastrepha ludens, is a polyphagous pest that is a constant threat to US agriculture because of its proximity to citrus production in border region of the United States and Mexico. Sterile Insect Technique has been used to manage and eradicate of the Mexican Fruit Fly. SIT, however, requires millions of sterile male flies to be released each week from mass-rearing facilities. The mass-reared colonies must deal with microbial infections that reduce survivorship, fecundity and male-male competition. Meta-transcriptomics joins culturing and next generation DNA sequencing techniques to understand these changing microbial communities. This study uses advances in microbial meta-transcriptome sequencing to analyze the microbial communities from the four major developmental stages of A. ludens. The composition of the microbial communities was analyzed and revealed the large transcriptional presence of the phyla Microsporidia, a known animal pathogen. Clustering and ordination analyses revealed groupings based on developmental phase for egg, larvae and pupa. Adult samples had little clustering; a not unexpected result given the known turn-over in microbial community that occurs in the first 5 d after pupation. The transcriptional activity of three known pathogens of A. ludens between developmental phases were investigated. Two of the three pathogens had significant transcriptional differences between the phases with the larvae phase having the most activity. This increase in transcriptional activity agrees with culture-based surveillance and current mitigation strategies employed in the mass-rearing facility. This result reinforces the utility of transcriptomics in combating microbial threats.

Insects and their associated microbiota have developed a sustained and mutually beneficial relationship, characterized by the influence of the symbiotic microorganisms on the host's physiological processes and fitness parameters. The Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), is one of the world's most ubiquitous, invasive, and harmful agricultural pests. In Tunisia, the medfly is widely distributed across all bioclimatic zones. However, in the absence of surveillance, infestations can escalate drastically, causing damage levels as high as 100%. Our study aimed to characterize the microbiome profile of Tunisian medfly populations from Zaghouan, Tozeur, Siliana, and Bizerte to understand the microbial dynamics implicated in the invasiveness and adaptability potential if SIT is applied. We conducted amplicon sequencing using MiSeq Illumina and a culture-dependent approach. Our findings revealed notable differences in symbiotic communities across regions. For instance, Serratia was prevalent in Tozeur populations, while Klebsiella showed high abundance in Bizerte. The composition of the bacterial communities within the medfly populations was influenced by several factors including the environmental conditions, geographical location, developmental stage, and the sex of the insects. Investigating the intricate relationship between insects and their microbiota is pivotal for understanding their biology and developing effective pest management strategies. Additionally, the isolation of bacteria from adult and larval medflies collected in the Bizerte region revealed the presence of bacterial species that could be utilized as attractants or supplements in larval artificial diets in the case of application of the SIT aiming at producing competitive sterile males.

The olive fruit fly (Bactrocera oleae) is a significant pest threatening olive production worldwide. Bactrocera oleae relies on symbiotic bacteria for nutrition, development, and adaptation to its environment. Among these, Candidatus Erwinia dacicola is the most dominant symbiont and plays a key role in the fly's physiology and ecological adaptation. Understanding the dynamics between B. oleae, Ca. E. dacicola, and other components of the B. oleae microbiome is essential for developing effective targeted area-wide pest management strategies. This study aims to leverage full 16S rRNA gene sequencing to enhance the characterization of microbiome diversity in wild B. oleae populations from different regions in Morocco: Ouezzane, Rabat, Tanger, Errachidia, and Beni-Mellal. The results revealed distinct microbiome compositions influenced by geographic locations, with Candidatus Erwinia dacicola as the dominant symbiont, followed by Erwinia persicina as a secondary contributor. Other bacterial taxa, including Asaia bogorensis, were also identified, highlighting the functional diversity within the olive fly microbiome. These findings provide insights into the microbial ecology of B. oleae, contributing to the development and enhancement of sustainable pest control strategies.

Enriching the diets of sterile fruit flies with bacterial species prior to their release has been shown to improve their survival and sexual competitiveness. However, most of the bacteria associated with fruit flies are enterobacteria, and some species have been classified as opportunistic pathogens. On the other hand, in diets that include hydrolyzed yeast, the effect of bacteria has been counterproductive, which could be due to the protein content in the diet being too high. Therefore, our aim here was to evaluate the effect of Lactobacillus casei, interacting with different contents of hydrolyzed yeast in the diet, on the performance of Anastrepha obliqua adults. In sexual competitiveness tests, the highest proportion of matings was achieved by males fed on a standard diet (3 : 1 sugar : yeast) plus L. casei. However, the differences with the 3 : 1 sugar : yeast diet was not significant. In survival tests, the 3 : 1 diet, without L. casei, showed the highest survival. The group fed with sugar plus L. casei showed the lowest survival. Females fed the 3 : 1 diet showed the highest fecundity, followed by those fed the 3 : 1 diet plus L. casei. Enriching the standard 3 : 1 diet with L. casei did not lead to significant improvements in male sexual performance, survival of either sex, or fecundity, compared to flies fed the standard diet alone. However, given that Lactobacillus is harmless to both humans and flies, further research into its potential application in the sterile insect technique (SIT) is recommended. Future studies should explore alternative formulations, varying concentrations, and other species within the Lactobacillus genus that may exert more pronounced effects on the biological traits of this fruit fly.

Glossina austeni Newstead (Diptera: Glossinidae) is a competent vector of the trypanosomes causing human African trypanosomiasis and the African animal trypanosomosis. Management of this pest has primarily involved trapping methods, Sterile Insect Technique, and research into vector competence–symbiotic interactions. Nevertheless, the use of entomopathogenic fungi (EPF) in integrated pest management programs for G. austeni control remains limited. Moreover, different tsetse fly species exhibit varying susceptibility to different EPF strains, indicating that no single strain is universally effective. Therefore, our study aimed to identify candidate EPF isolates for G. austeni management, evaluate the effects of temperature on the radial growth of these potent isolates, and assess the impact of the candidate EPF on the gut microbiome of G. austeni. Consequently, 16 Metarhizium anisopliae (Metschn.) Sorokin isolates were screened against G. austeni using dry conidia in an infection chamber, with the most virulent isolates having LT₅₀ values of 3.95−9.37 d. Temperature significantly influenced the radial growth, conidia germination, and yield of these strains. There were also significant differences in conidia acquisition, retention and transmission between male and female G. austeni flies. Furthermore, all conidia receivers carried sufficient conidia, 5 d post-interaction with EPF-challenged conidia donors. Microbiome analysis revealed Wigglesworthia, Serratia, Klebsiella, and Escherichia as the most abundant taxa. Among the M. anisopliae isolates, ICIPE 82 exhibited the fastest radial growth and highest thermostability, hence selected as a potential biopesticide candidate for managing G. austeni. This study demonstrates the efficacy and potential of M. anisopliae ICIPE 82 as a biopesticide for controlling G. austeni.

Sterile Insect Technique (SIT) has proven effective to reduce tsetse population density in large infected areas where animal African trypanosomosis (AAT) and human African trypanosomiasis (HAT) elimination was difficult to achieve. However, the decrease in mass production of insectary-reared tsetse and the limited but incomplete knowledge on symbiont–trypanosome interaction over time, impede large-scale use of SIT. We investigated the spatiotemporal changes in symbiont prevalence and symbiont–trypanosome interactions in wild tsetse of Sora-Mboum AAT focus in northern Cameroon, collected in 2019 and 2020, to provide insights into the mass production of refractory tsetse. Spiroplasma spp., Sodalis glossinidius and trypanosomes were screened with PCR. G. tachinoides was the most abundant Glossina species found in Sora-Mboum focus. Symbiont prevalences in G. tachinoides were higher in 2019 compared to 2020, from 67.6% to 53.5% for Spiroplasma spp. and from 28.8% to 8.1% for S. glossinidius. These symbionts were also found at higher prevalence in flies from Mouhoun HAT focus in Burkina Faso. Four trypanosome taxa (Trypanosoma congolense forest type, T. congolense savannah type, T. brucei s.l., and T. vivax) were found in Sora-Mboum focus and Mouhoun focus, though at lower prevalence in Mouhoun. The presence of Spiroplasma spp. in adult tsetse was negatively associated with that of trypanosomes. Our study highlights the potential of Spiroplasma spp. as a good paratransgenesis candidate to enhance SIT application. This symbiont is naturally found in high proportions of tsetse and could prevent factory flies from acquiring and transmitting trypanosomes during their lifespan when released for population density control.

Tsetse flies are insects of significant public health and zoonotic importance as they are the main vectors of African trypanosomes. To date, an effective vaccine is unavailable and efforts to limit the spread of the disease primarily rely on controlling the tsetse populations. The discovery of Spiroplasma (class Mollicutes) in Glossina fuscipes fuscipes (Gff) (palpalis subgroup), offers promising insights into its potential use as a biological control agent to hinder trypanosomes infection in tsetse flies. Indeed, a negative correlation between Spiroplasma and trypanosome co-infection has been observed. Using a laboratory strain of Gff, we provide fundamental biological insights into the effects of Spiroplasma infection on the mating behavior of the fly. We found a sex-biased Spiroplasma infection, with males exhibiting a higher infection rate. Mass mating experiments revealed a higher mating propensity in Spiroplasma-infected flies. Additionally, the presence of Spiroplasma influenced premating isolation, leading to nonrandom mating patterns that favored the pairing of individuals with matching infection statuses. Moreover, we present evidence of Spiroplasma vertical paternal transmission. By analyzing female reproductive tissues at 2 and 24 h postmating, we confirmed that an infected male can transfer Spiroplasma to the female via the spermatophore, which can subsequently migrate to the spermathecae. This study provides foundational insights into the role of Spiroplasma in tsetse fly mating behavior and provides supporting evidence for vertical transmission from infected males.

The Mediterranean fruit fly is an agricultural pest of a wide variety of fruit crops. An effective method to counteract them in the field is through the application of the sterile insect technique, which requires the mass-production of sterile males. The presence of pathogens, and specifically viruses, threatens the well-being of mass-reared insects generating an interest on the development of strategies for viral elimination or containment. Thirteen RNA viruses have been described in the medfly although so far only one of them, Ceratitis capitata nora virus, has been associated with detrimental effects on medfly development. In this context, medfly larvae were supplied with a chemical compound (formaldehyde) and an antiviral compound (ribavirin) via oral feeding to (1) test the potential of these compounds for viral elimination and (2) analyze their effect on medfly development. Overall, formaldehyde treatment did not reduce the viral titer for any of the tested viruses, while ribavirin effectively reduced the levels of two widespread RNA viruses but not in a dose–response manner. However, the addition of both compounds correlated with detrimental effects on medfly fitness, arguing against their use in mass-rearing facilities.

Trypanosomiasis, transmitted by tsetse flies (Glossina spp.), poses a significant health threat in 36 sub-Saharan African countries. Current control methods targeting tsetse flies, while effective, allow reinfestation. This study investigates paratransgenesis, a novel strategy to engineer symbiotic bacteria in tsetse flies, Sodalis glossinidius, to deliver anti-trypanosome compounds. Disrupting the trypanosome life cycle within the fly and reducing parasite transmission could offer a sustainable solution for trypanosomiasis control. In this context, we tested the effect of cecropin, reported to be lethal for Trypanosoma cruzi (Chagas disease) and TbgTCTP (Translationally Controlled Tumor Protein from Trypanosoma brucei gambiense), previously reported to modulate the growth of bacteria isolated from the fly microbiome, to delay the first peak of parasitemia and the death of trypanosome-infected mice. We have successfully cloned and transfected the genes encoding the two proteins into Sodalis strains. These Sodalis recombinant strains (recSodalisTbgTCTP and recSodaliscecropin) have been then microinjected into the L3 larval stage of Glossina palpalis gambiensis flies. The stability of the cloned genes was checked up to the 20th day after microinjection of recSodalis. The rate of fly emergence from untreated pupae was 95%; it was reduced by nearly 50% due to the mechanical injury caused by microinjection. It decreased to nearly 7% when larvae were injected with recSodalisTbgTCTP, which suggests TCTP could have a lethal impact to larvae development. When challenged with T. brucei gambiense, a slightly lower, but statistically non-significant, infection rate was recorded in flies harboring recSodaliscecropin compared to control flies. The effect of recSodalisTbgTCTP could not be measured due to the very low rate of fly emergence after corresponding treatment of the larvae. The results do not allow to conclude on the effect of cecropin or TCTP, delivered by para-transgenesis into the fly's gut, on the fly infection by the trypanosome. Nevertheless, the results are encouraging insofar as the technical approach works on the couple G. p. gambiensis/T. brucei gambiense. The next step will be to optimize the system and test other targets chosen among the ESPs (Excreted-Secreted Proteins) of the trypanosome secretum, or the differentially expressed genes associated with the sensitivity/resistance of the fly to trypanosome infection.

Tsetse flies (Glossina spp.) can vector the parasites (Trypanosoma spp.) that cause the socioeconomically devastating neglected tropical diseases human and animal African trypanosomoses. In addition to this parasite, tsetse can harbor four genera of endosymbiotic bacteria, including Wigglesworthia, Sodalis, Wolbachia, and Spiroplasma, which are functionally crucial for the fly's physiological homeostasis and/or are potentially useful for the development of disease control strategies. Recent discoveries indicate that Spiroplasma infection negatively impacts tsetse fecundity. Conversely, housing the bacterium can benefit its fly host by making it unusually refractory to infection with parasitic African trypanosomes. In this study, we assessed the physiological impact of Spiroplasma infection on a laboratory colony of Glossina fuscipes fuscipes (Gff). For this purpose, two distinct Gff colonies were established: a Spi– colony that harbors a low Spiroplasma infection prevalence and a Spi+ colony that harbors a high Spiroplasma infection prevalence. Fitness parameters for both colonies revealed no significant differences in the length of larval development, adult eclosion rate, and flight propensity. However, flies from the Spi+ colony presented with lower fecundity and higher overall mortality than did individuals from the Spi– colony. Furthermore, males from the Spi– colony exhibited a competitive mating advantage over their Spi+ counterparts in a field cage setting. These findings have potential implications for the improvement of mass-rearing of Gff for sterile insect technique (SIT) applications.

The High Biosecurity House of the Mediterranean Fly in Metapa (CABIM3, in its Spanish acronym) is a space within the new Moscamed Mexico facility designed to select favorable traits that mitigate the negative effects of mass production while enhancing male competitiveness, ultimately increasing the efficiency of the sterile insect technique (SIT). The CABIM3 serves as an environmentally enriched space where insects, whose offspring will initiate the mass production process, are confined. In this study, the sexual performance of males and oviposition behavior of females from mass-reared and wild strains of Anastrepha ludens (Loew) and A. obliqua (Macquart) were compared under field cage conditions, inside CABIM3 and in an orchard, to evaluate the suitability of CABIM3's environmental conditions, including light intensity, temperature, and relative humidity. The results revealed significant differences in male sexual performance and female oviposition behavior between the CABIM3 areas and the orchard. Despite these differences, our findings suggest that a breeding facility like CABIM3 could be a valuable tool for improving the attributes of insects used in SIT programs.

Anastrepha ludens (Loew) is controlled in Mexico using sterile insect technique (SIT). SIT relies primarily on mass-reared insects, which are subjected to the effects of selection during colonization and rearing, and that frequently result in modifications of their biology and behavior. Here, we propose and evaluate a novel “bi-environmental cage” for colony management which promotes more natural sexual selection. The cage allows each sex to reach sexual maturity in separate compartments acclimatized according to natural conditions. Females mature in areas where they can recognize oviposition sites, while males mature in areas populated with small trees to allow establishment of territories in leks and performance of courtship behaviors. To determine whether the bi-environmental cage can minimize the potential adverse effects on mating competitiveness, two strains of A. ludens were tested; wild flies strain and genetic sexing strain Tapachula 7 (mass-reared flies). We found that after 4 generations in the mass-reared flies in the bi-environmental cage showed a level of fecundity similar to that of flies from the conventional cage. A similar pattern was also seen in the case of wild flies in both types of cages. In addition, other biological attributes of the wild strain assessed over six generations showed adaptability to mass-rearing conditions. Wild males from the bi-environmental cages were more sexually competitive than those from the conventional cage. Our results show that it is possible to mitigate many of the detrimental effects of domestication on the sexual performance of mass-reared males by using close-to-natural conditions for colony management.

The sterile insect technique (SIT) for tsetse involves releasing sterilized males to outcompete wild males in mating, resulting in nonviable progeny. Balancing optimum sterility and male quality is crucial. While irradiation in hypoxia or anoxia is routine for tephritid flies, its effect on tsetse, especially postrelease, is not well understood. We conducted experiments to understand the impact of irradiation in hypoxia on the sterility of the F1 generation of Glossina morsitans morsitans (Gmm). Initially, we tested the impact of 1-h preconditioning in hypoxia before irradiation and continued hypoxia up to 24, 48, and 72 h postirradiation on the emergence and flight propensity of treated males. We then assessed mating ability, survival, pupae per initial female (PPIF) and residual fertility of flies following irradiation at various doses after 1-h hypoxia conditioning. Finally, we determined the PPIF and residual fertility of the F1generation and the emergence of treated flies to the F2 generation. Results show that short-term conditioning (1 h) in hypoxia maintains or improves the qualities of the irradiated Gmm pupae. Regardless of irradiation in normoxia or hypoxia, higher residual fertility was observed in females than males in the F0 generation, and higher residual fertility in the F1 than the F0 generation for both sexes. However, the emergence rates of males decreased in the F2 generation compared with the F1 generation, a noteworthy finding for SIT programs for tsetse, implying diminishing populations of sterile flies.