Lepidopteran species are amongst the most damaging pests of food and fiber crops world-wide. Pest lepidopterans are often managed injudiciously by spraying crops with large amounts of broad-spectrum—and often—persistent insecticides. In view of increased occurrence of resistance against these insecticides, and their negative impacts on the environment and ecosystems, the need for control tactics that are not only effective but also friendlier to the environment is becoming more and more pressing. Both the sterile insect technique (SIT), and the related inherited sterility (IS) technique offer great potential as additional control tactics for integration with other control methods in area-wide integrated pest management approaches against lepidopteran pests. However the SIT/IS can only be applied successfully when the released sterile insects can effectively compete with their wild counterparts for mating with wild females. Although there are a number of programs where the SIT has been used very effectively against key lepidopteran pests, there is great potential for further expansion of the SIT/IS technology to target other key lepidopteran pests, or to improve the SIT/IS for already targeted Lepidoptera. Such expansion could be facilitated through improvements of mass-rearing, measurement and control of quality, handling, irradiation, shipping, release and field assessment technologies that would increase program efficiency and efficacy. To foster such advances, the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, implemented a Coordinated Research Project (CRP) from 2008 to 2014 entitled “Increasing the efficiency of Lepidoptera SIT by enhanced quality control”. This Project organized and sponsored research and development on the following key objectives: (1) identify and investigate factors and variables affecting the quality of the produced and released insects and their field performance, (2) identify and develop new tools and methods to assess and predict the field performance of sterile insects, and (3) improve the artificial rearing of several moth species through a better understanding and management of genetic resources. The key research outputs of the CRP are summarized in this paper.

A user-friendly simulation tool for determining the impact of the sterile insect technique/inherited sterility technique (SIT/IS) on populations of the African sugarcane stalk borer, Eldana saccharina Walker (Lepidoptera: Pyralidae) is described in this paper. The simulation tool is based on a spatio-temporal model. The design of the simulation tool is such that it is applicable for use in a number of pest/crop and pest control scenarios. It uses 4 interacting subsystems (pest species population dynamics, crop dynamics, environmental dynamics and economics) within a specified spatial domain. Furthermore, the spatial domain describes the layout of the agricultural crop (position, size, shape, crop age and variety of the different fields contained within the crop area). The pest species population subsystem describes E. saccharina population dynamics (but is designed to also include population dynamics of other pest species) under the influence of the IS technique. The E. saccharina module developed utilizes mean-field and spatio-temporal models, and includes dynamics of all E. saccharina life stages under the influence of the control measure. Only temperature and damage caused by E. saccharina are currently included as variables in the sugarcane dynamics subsystem. This subsystem estimates stalk length as a function of time and temperature, and sucrose percentage as a function of damage caused by E. saccharina boring. Interaction between E. saccharina population growth and sugarcane growth is described by a decreasing s-shaped density-dependent mortality function—the older the cane, the higher the carrying capacity (more food resources) and corresponding infestation and damage levels. The only environmental factor considered as an independent variable in the environmental dynamics subsystem is temperature. Possible extensions to this subsystem are discussed. The economics subsystem developed includes the estimation of the recoverable value, percentage, expected revenue and the cost of control. No other farm expenditures are taken into account. As such only profit or loss expected from applying the IS technique is estimated. The profit or loss is defined as the increase in revenue expected less the cost of applying a pest control measure. An example of using the simulation tool is presented in the context of a real field scenario of a simulated SIT/IS program against E. saccharina at a pilot site near the Eston area of KwaZulu-Natal, South Africa.

Codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), a highly invasive species, recently became established in the Hexi Corridor, which is a long narrow passage area with many oases surrounded by deserts and tall mountains in Gansu province, China. The corridor is an important temperate fruit growing region in northwestern China as well as a natural barrier to prevent C. pomonella from invading other fruit growing areas of the country. Since the codling moth was firstly reported, pome fruit damage in this corridor has been severe. The sterile insect technique (SIT) is considered a possible effective control tactic for integration in a future area-wide integrated pest management (AW-IPM) program against C. pomonella in the corridor. Knowledge of population genetics and more specifically of genetic differentiation and gene flow patterns may be important for developing AW-IPM strategies that include the SIT. In the current study, we collected C. pomonella samples from 8 populations distributed across 2 adjacent regions in the Hexi corridor that are geographically separated by stone deserts and high mountains. Eight microsatellite loci were used to investigate the genetic diversity, structure and differentiation of these 8 populations. Significant genetic differentiation was found between populations of each of the 2 regions, whereas populations within each region showed a similar genetic structure, demonstrated by higher Nm and lower FST values for population pairs within the same region than in pairs between the regions. Our findings indicate limited gene flow of C. pomonella between the 2 regions, which suggests that SIT can be implemented to control the pest in the Hexi Corridor of China.

Eldana saccharina Walker (Lepidoptera: Pyralidae: Gallerinae) occurs on many graminaceous crops and several wild grasses and sedges throughout Africa. It has been reared at the South African Sugarcane Research Institute (SASRI) since the 1970s to study its biology and behavior, as a host for natural enemies and to provide insect material for the plant breeding program. Studies were completed on laboratory-reared E. saccharina of South African origin to assess fecundity, fertility and male and female mating frequencies. Mean fecundity of E. saccharina was 518 ± 27.5 (mean ± SE) eggs per female, up to a maximum of 798 eggs. Mean egg hatch (fertility) of E. saccharina was 63.2 ± 4.2%. In the laboratory, 56.7% of E. saccharina females mated only once but on average females mated 1.5 ± 0.1 times (maximum of 3). Males mated with a maximum of 6 females per male but on average males mated 3.3 ± 0.7 females. Most matings (93%) occurred on the first and second nights after male emergence, and the females oviposited most of their eggs (49.9 ± 3.9%) on the second night after emergence. Eldana saccharina's high fecundity confirmed its potential as a crop pest. This study has, for the first time, confirmed that male and female E. saccharina were able to mate more than once under controlled laboratory conditions. This has important implications for calculating required release rates of sterilized males to obtain adequate sterile to wild male over-flooding ratios in area-wide integrated pest management programs that have a SIT component.

Laboratory-reared male and female Eldana saccharina Walker (Lepidoptera: Pyralidae: Gallerinae)—a major pest of sugarcane (Saccharum spp.; Poales: Poaceae) in South Africa—were exposed to increasing doses of γ-radiation to assess this species' suitability for the sterile insect technique as part of an area-wide integrated pest management (AW-IPM) program. Irradiated male and female moths were crossed with non-irradiated counterparts and irradiated counterparts at the same radiation dose. Male and female moths were treated with radiation doses of 150, 200, 250, 300 and 350 Gy, and the following crosses were made: irradiated males (T♂) mated with untreated females (U♀) and irradiated females (T♀) mated with untreated males (U♂). Males and females of the T♀ × T♂ cross were exposed to the same radiation doses except in the 350 Gy treatment. Fertility (egg hatch) declined significantly with increasing doses of radiation in all crosses. Female progeny from the T♀ × U♂ and T♀ × T♂ crosses were more sensitive to irradiation than female progeny from the U♀ × U♂ and U♀ × T♂ crosses and were completely sterile when treated with either 200 Gy or 150 Gy. Treated males mated with untreated females still had a residual fertility of 0.19% when exposed to 350 Gy of radiation. The fertility of E. saccharina is therefore sensitive to increasing doses of ionizing radiation and this species is a suitable candidate for further development of the SIT as a component of an AW-IPM program. The fecundity of untreated E. saccharina females mated with irradiated males was not affected by radiation. The availability of a great number of infertile eggs can be regarded as a benefit in a program where natural enemies are combined with the SIT because non-fertile eggs can provide additional hosts for egg parasitoids and be a food source for predators.

The tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is one of the most important pests of tomato. With the purpose of developing environmentally friendly control tactics such as the inherited sterility (IS) technique against this species, it is essential to understand its genetics and biology. In this paper we analyzed the karyotype, sperm morphology and sperm ratio in wild-type and X-ray irradiated individuals of T. absoluta. The diploid chromosome number of T. absoluta was 2n = 58 including the pair of sex chromosomes: ZZ in males and WZ in females, which were the largest elements of the complement. Irradiation of pupae in an X-ray machine with a dose of 200 Gy generated various types of chromosomal rearrangements including translocations and fragmentations, resulting in altered chromosome numbers. The analysis of spermatozoa in T. absoluta revealed a significant morphological difference between apyrene and eupyrene sperm bundles. Irradiation with X-ray doses of 100, 150, 200 and 250 Gy did not have a significant effect on the apyrene to eupyrene sperm ratio. However, males irradiated with 300 Gy produced significantly more apyrene sperm than non-irradiated males. All the doses applied influenced the morphology of eupyrene sperm bundles. The modified eupyrene sperm bundles could be used as a bioindicator during the monitoring of an IS program after the release of irradiated males. We found that the modified eupyrene spermatozoa were transferred to the bursae copulatrices of the females. Males treated with 200 Gy transferred a greater proportion of modified eupyrene sperm than untreated males. The results presented herein provide essential information on the cytology of T. absoluta, which is required to evaluate the quality of the released insects, and for better understanding and application of IS against this economically important pest.

The efficacy of the sterile insect technique (SIT) applied as part of area-wide integrated pest management (AW-IPM) depends on efficient transfer of sperm carrying dominant lethal mutations from sterile males to wild females. Success or failure of this strategy is therefore critically dependent on quality and ability of sterile males to search for and copulate with wild females. The African sugarcane borer, Eldana saccharina Walker (Lepidoptera: Pyralidae) is an economic pest of sugarcane targeted for control in South Africa using an AW-IPM approach with a SIT component. As part of further steps towards development of the technique, levels of mating competitiveness and compatibility were assessed by observing the extent to which individuals from different populations interbreed when confined together under both laboratory and semi-field conditions. Three types of pair-wise competition experiments were conducted: non-irradiated laboratory adults vs. non-irradiated wild adults, irradiated (200 Gy) laboratory adults vs. non-irradiated wild adults, and non-irradiated laboratory adults vs. irradiated (200 Gy) laboratory adults. Data from these tests were used to generate indices for mating performance and measuring sexual compatibility between strains. Irrespective of trial location, wild moths did not discriminate against irradiated or laboratory-reared moths, indicating no negative effects on acceptability for mating due to laboratory rearing or radiation treatment. In general, irradiated males mated significantly more than their wild counterparts regardless of the type of female, which indicated that they were still as competitive as their wild counterparts. The mating indices generated showed no evidence of incipient pre-mating isolation barriers or sexual incompatibility with the wild strain. Data presented in this paper therefore indicate that there is scope for further development of the SIT as an addition to the arsenal of tactics available for AW-IPM of this economic pest.

Male and female litchi stem-end borers, Conopomorpha sinensis (Lepidoptera: Gracillariidae) were γ-irradiated in a 60Co source during several periods of their pupal development and as newly emerged adults. When mature pupae, the most suitable stage for irradiation, received increasing γ-radiation doses, the emergence rate, flight ability and adult longevity were increasingly diminished. Females that emerged from mature pupae irradiated with 200, 250, and 300 Gy did not oviposit any eggs when mated with either non-irradiated males (UM) or treated males (TM), indicating that 200 Gy was a sufficient dose for inducing complete sterility in females. The hatch rates of eggs oviposited by non-irradiated females (UF) mated with males irradiated with either 200, 250 or 300 Gy were 31, 13.5 and 0.67%, respectively. However, if the father—parental generation (P)—of the male in the cross, UF × F₁ M, had been irradiated with either 200, 250 or 300 Gy, then the percent hatch of the eggs produced was either 9.37 ± 1.68, 0 or 0%, respectively; which indicated that 250 and 300 Gy applied to P generation males resulted in complete sterility in F₁ males. The sexual competitiveness (C) of male litchi stem-end borers that had been irradiated with 250 Gy ranged between 0.53 and 0.62 as measured by ratio tests in laboratory cages with UF:UM:TM ratios of 1:1:1, 1:1:3 and 1:1:5. In a field cage experiment, a mean C value of 0.48 was observed when males were treated with 250 Gy. The C values obtained suggest that males treated with 250 Gy could compete adequately with wild litchi stem-end borers both under laboratory and field cage conditions to warrant pilot scale field tests.

Flight abilities and dispersal distances of males of irradiated vs. untreated European grapevine moths (Lobesia botrana [Denis & Schiffermüller]: Lepidoptera: Tortricidae) were assessed in a flight assessment cage and in a vineyard. Newly emerged adult male moths were either untreated or γ-irradiated either with 150 Gy or 350 Gy, and each group was marked with a different colored fluorescent dust. Males were released in a laboratory flight assessment cage (70 × 40 × 50 cm) and at the center of a vineyard. The flight assessment cage test revealed significant differences in the flight responses of irradiated and untreated L. botrana males to calling females during the first 2 days after the initiation of the test. The greatest percentage of non-flying males (47%) was observed in the 350 Gy-treatment, whereas no significant differences were detected in male flight ability between untreated and 150 Gy γ-irradiated male moths. Six hundred male moths were released in a vineyard with a rectangular trapping grid around a central release point, and traps were baited with a synthetic pheromone. One hundred and thirty one males (21.8%) were recaptured, with the farthest being caught 40 m from the release point. No differences were observed in male field performance between 150 Gy γ-irradiated and untreated male moths, whereas 350 Gy γ-irradiated males showed limited field dispersal when compared with either 150 Gy γ-irradiated or untreated male moths. The results, the values of several attributes (flight ability, dispersal distance and recapture rate of released males in pheromone-baited traps)—which are critical for effective population suppression by the sterile insect technique with inherited or F₁ sterility (SIT/F1)—were significantly decreased by increasing the radiation dose applied to L. botrana males from 150 Gy up to 350 Gy. The flight assessment cage proved to be a valuable tool for measuring differences in the quality of untreated and irradiated moths.

Assessment of quality of male insects has been done primarily in area-wide integrated pest management (AW-IPM) programs that have a sterile insect technique (SIT) component. Routine monitoring of sterile male quality needs to be carried out both in the mass-rearing facility and in the field. Simple bioassays, which can be conducted in the laboratory or under semi-field conditions, would be potential surrogates for laborious field tests that are usually very costly. In the laboratory, a flight mill system was used to assess the quality of males of the litchi stem-end borer, Conopomorpha sinensis (Lepidoptera: Gracillariidae), in terms of flight distance, flight duration and speed. Flight distance, duration, mean speed and greatest speed of non-irradiated adult males were 13,926 m, 29,365 s, 0.42 m/s, and 1.01 m/s, respectively, during a 24 h period. Although the values of these parameters of non-irradiated males were greater than corresponding values for moths irradiated with 150 and 200 Gy, there were no significant differences between the various treatments and the non-irradiated control group. These data suggest that irradiation with 150 and 200 Gy did not significantly affect the flight ability of male litchi stem-end borers. This study also included 2 field release and recapture experiments. The data of the field experiments indicated that recapture rates, dispersal distances, and dispersal directions of the 150 and 200 Gy irradiated males were not significantly different from those of non-irradiated males. These data indicate that the ability of litchi stem-end borer males irradiated with doses of 150 and 200 Gy to disperse in the field was not impaired in comparison with non-irradiated males.

Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is one of the most devastating pests of tomato. We studied whether partially sterile T. absoluta males and fully sterile females were capable of suppressing wild populations of this moth in semi-controlled conditions. After irradiating T. absoluta pupae with 200 Gy, emerged males and females were released at 10:1 (treated:untreated) and 15:1 over-flooding ratios inside field cages containing tomato plants. The number of eggs and larvae produced was recorded once per wk during 3 mo. An over-flooding ratio of 10:1 caused a decline in larvae production compared with the untreated control cages, but these differences were not statistically significant. Using an over-flooding ratio of 15:1, the moth population in the cages with irradiated insects decreased significantly compared with those in the untreated control cages. In addition, the possibility of combining inherited sterility and a natural enemy as a strategy to manage this pest was investigated. We studied in no choice and choice tests the predation behavior of females of the egg predator Tupiocoris cucurbitaceus (Hemiptera: Miridae) on eggs derived from the following parental T. absoluta crosses: ♀_U × ♂_U (control), ♀_U × ♂_I and ♀_I × ♂_U (where U = untreated, I = irradiated). In the no choice test, females of the T. cucurbitaceus readily consumed T. absoluta eggs regardless of their origin. In the choice test, T. cucurbitaceus females consumed similar numbers of eggs of untreated parents and eggs oviposited from crosses in which the male had been irradiated. However, the mirid females preyed on significantly more eggs—roughly 20% more—from irradiated females than on eggs from untreated females. We conclude that it is possible to achieve a certain level of suppression of a T. absoluta wild population through the release of irradiated insects, and that it is technically feasible to combine the use of the predator T. cucurbitaceus with inherited sterility to control this moth pest.

One of the most important components of a program that has a sterile insect technique (SIT) component is an appropriate irradiation dose. Knowing the organismal dose-response enables the selection of a dose that induces the highest level of sterility while preserving the sexual competitiveness and other desired qualities of the sterile insect. Finding this balance in Lepidoptera is crucial because of the use of inherited (F1) sterility, where the irradiated parent must be competitive enough to mate while its offspring must be sterile. Manipulations of atmospheric oxygen content have been shown to be an effective way of lowering post-irradiation somatic damage while preserving sterility and improving sterile insect performance, particularly in fruit flies. In this study we tested the irradiation dose response of adults of the cactus moth, Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), and the effects of those doses on sterility, longevity, and F₁ performance, and whether a nitrogen conditioning-treatment (anoxia) prior to and during irradiation affected these metrics. We found that male and female fecundities were not impacted by dose or atmospheric treatment, but females were sterilized at lower doses than males. Eggs of irradiated parents took longer to hatch than those of unirradiated controls, and offspring of moths irradiated in anoxia lived longer in the absence of food and water. Anoxia conditioning rescued female fertility at intermediate doses but had no similar rescue effect on male fertility, which was always greater than female fertility at a given dose. Males generally lived longer than females and anoxia had a strong effect in lowering the male mortality rate and extending lifespan at a given dose. We show evidence that anoxia-conditioning prior to and during irradiation as part of a lepidopteran program with an SIT component could improve parental and larval performance and longevity.

Sperm behavior represents one of the attributes in the radiogenetic technique called inherited sterility crucial for its effectiveness to suppress populations of Spodoptera litura (F.) (Lepidoptera: Noctuidae). Quantitative and qualitative assessments of S. litura sperm behavior (production, descent, activation, movement and transfer to females) were made in parental (P) males that received sub-sterilizing irradiation doses of either 100 or 130 Gy and in their F₁ generation males. Age dependent production of eupyrene and apyrene spermatozoa in the testes were not affected by such irradiation in the P males, nonetheless a slight but significant effect occurred in the F₁ generation. During the rhythmic cycles of sperm descent in the photophase and scotophase, the profile and proportion of sperm descent from the testes to the upper vas deferens (UVD), seminal vesicle (SV) and duplex were not significantly influenced by irradiation in P and F₁ males. Sperm activation—assessed as percent active apyrene sperm and their intensity of motility—was not diminished in irradiated P males, while in F₁ males it showed a slight but significant reduction. Mating status was not a markedly pronounced factor in eliciting the motility of irradiated sperm. Sub-sterilized P males and their F₁ progeny were nearly as competitive as non-irradiated males in terms of sperm transfer from male to female. Successful amphimixis occurred between the altered genomes of either irradiated P males or F₁ males and the non-irradiated female genome; consequently dose dependent reductions in percent egg hatch were observed in the hatching of F₁ and F2 eggs. Irradiation with either 100 or 130 Gy did not adversely influence the sperm characteristics in the irradiated P males and their F₁ male progeny, and this study validated the sperm' viability and performance in irradiated P males and of their F₁ sons. The findings indicated that these genetically altered sperm would fertilize the eggs of wild females and lead to effective control of this tropical pest.

The dose of radiation applied to the Oriental leafworm (also known as the common cutworm), Spodoptera litura (F.) (Lepidoptera: Noctuidae) for use in inherited sterility (IS) sterility programs must not materially reduce the quality and competitiveness of the released males. To assess the quality of sub-sterilized male moths and their F₁ progeny, their flight and mating behaviors were investigated. Thus, parental (P) generation S. litura males that had been irradiated with either 100 or 130 Gy and their F₁ male descendants were subjected to flight assay tests to assess flight ability, orientation towards a pheromone source and mating behavior, i.e., mating performance using various sex ratios, sequential matings, and effect of female age. The orientation behavior of either P or F₁ males towards pheromone-baited traps was not different from that of non-irradiated males. Mating abilities of irradiated P generation males and their F₁ male progeny with non-irradiated females were studied using several sex ratios, but large differences were not found even when the sex ratio was increased from 1:1 to 5:1. A study on sperm use patterns revealed the precedence of sperm of the last male to mate, for example, when a non-irradiated female was sequentially mated with a non-irradiated P male, followed by an irradiated P male and finally by an F₁ male, as well as in all other possible sequences of such males. Mating success, remating propensity and fertility were significantly influenced by mating sequences that included irradiated males. Further, the age of the female at the first mating influenced fertility in matings with F₁ males, and mating success and remating propensity were reduced in several of sequences of matings involving non-irradiated and irradiated males and F1 males, with reductions being more apparent when females were aged 5–6 d. The present study indicates that gamma doses of either 100 or 130 Gy are suitable for the suppression of S. litura using an IS approach.

Irradiation is used to provide sterile insects from mass-rearing facilities, but irradiation can degrade insect quality. A system is described that uses repeatable pheromone stimuli to activate male moths housed with clean airflow in a commercially available insect activity meter, for potential use in quality assessment of mass-reared moths in sterile insect programs. We tested sexually mature wild and sterile light brown apple moth (LBAM), Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), males at the commencement of scotophase in a simple assay for increased mate-seeking activity after a 2 s stimulus with a 4-component synthetic pheromone source for 2 s, after a 5 s baseline of activity. Male activity at the upwind end of the glass tube was assessed by infrared beam. Next, we tested if a temperature shock at 30 °C in the pupal stage for various durations (0, 1, 2 or 4 h) would have an effect on male moth response to the sex pheromone. The results indicated similar baseline activity in clean airflow, but a significantly greater response after pheromone stimulation from non-irradiated males, compared with irradiated males. Responses from irradiated males averaged 78% of the control response (n = 320). The temperature shock did not appear to change the response of the non-irradiated moths (n = 64), but there was a slight decline in response by irradiated moths at the 2 and 4 h temperature shock durations. The system could be readily modified to be suitable for factory scale quality assurance.

The Australian light brown apple moth (LBAM) (Epiphyas postvittana) (Walker) (Lepidoptera: Tortricidae) is a pest in Australia, New Zealand and now California (USA). The use of sterile insects in combination with mating disruption and biological insecticides has the potential to eradicate outbreaks in urban areas. The sexual competitiveness of irradiated insects is an important component of the effectiveness of the sterile insect technique (SIT), but standard techniques to measure the sexual competitiveness have been developed only for irradiated tephritid fruit flies. In particular, field cage trials have been used to measure the compatibility and competitiveness of irradiated fruit flies in comparison with wild fruit flies. Trials were carried out to determine if such tests could be adapted for a moth species. Parameters of quality or competitiveness evaluated were the proportion of the moths that mated, relative sterility index, index of sexual isolation, and mating competitiveness based on the egg hatch in the various crosses. Results showed that with the release of sterile moths of both sexes (bisex) there was little difference in competitiveness—expressed as the Relative Sterility Index (RSI)—between moths irradiated at 200, 250 and 300 Gy (irradiated either in the pupal or adult stages), but if a Fried competitiveness test was used to generate competitive C values then greater competitiveness was found at the lower doses of irradiation, but this difference was not statistically significant. Modified test procedures were developed in which the moths in field cages—after having had sufficient opportunity to mate—were egged individually and dissected to determine the presence of 1 or more spermatophores; then egg sterility and spermatophore presence were used to determine the mating type, e.g., wild female × irradiated laboratory male, etc. Results indicated that sterile-male-only releases have the potential to increase mating competitiveness of the released irradiated moths, but this conclusion requires additional experiments for confirmation.

Plant chemical signals are important olfactory cues for the survival and reproduction of phytophagous insects. The diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) is a Brassica spp. (Brassicales: Brassicaceae) specialist pest, with most of its life events occurring on Brassica spp. hosts. We conducted a scanning electron microscopy study on the morphology and distribution of antennal sensilla of male and female P. xylostella. Seven morphological types of sensilla were identified in the antennae of P. xylostella: 3 types of sensilla trichodea (Tr I, Tr II and Tr III), sensilla chaetica, sensilla coeloconica, sensilla auricillica and sensilla styloconica. One particular type of trichoid sensillum (Tr III) was present only in the males. The presence of numerous pores or deep longitudinal grooves on the surfaces of 5 morphological types of sensilla indicated that their major function is olfactory. Single sensillum recordings were also carried out on the trichoid sensilla of the female diamondback moth to identify the olfactory receptor neurons (ORNs) and to determine the response spectra of the ORNs, using a panel of 39 host and non-host volatile compounds. Based on the response profiles, 42 responsive trichoid sensilla could be segregated into 4 sensillum classes. Each sensillum appeared to contain 3 co-compartmentalized ORNs, and therefore a total of 12 classes of ORNs were identified from these sensilla. Each ORN class showed a narrow response spectrum, with some ORNs specialized for green leaf volatiles and (±)-linalool that are present in brassicaceous hosts, while several other ORNs responded to 2 non-host volatile sesquiterpenes, (E)-β-farnesene and germacrene D, as well as (E)-β-caryophyllene, a host-related sesquiterpene volatile. The sensitivity and selectivity of the female diamondback moth towards certain host plant volatiles warrants further investigation for potential behavioral manipulation to control this pest.

Knowledge of host chemical volatiles has tremendously informed the fundamental understanding of plant-insect interactions as well as revealed opportunities for the creative use of plant-derived chemicals in the food and flavor industries and in insect pest management. This study was undertaken to assess the host searching behaviors of diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae) females in relation to their mating status and conspecific larval herbivory. This involved measurement of odor-modulated upwind flight of female moths in a wind tunnel. Subsequently, the host location, recognition and acceptance behaviors of gravid females in relation to either intact uninfested or larvae-infested cabbage, Brassica oleracea L. (capitata group; Brassicales: Brassicaceae) hosts were compared in no-choice and dual-choice tests. Mating status had little effect on the female responses to host odor and flight duration. However, female moths were significantly more attracted to conspecific larvae-infested cabbage plants and had significantly shorter flights than in relation to intact uninfested cabbage hosts. Behavioral responses of gravid females were consistent when offered intact and larvae-infested cabbage hosts in both no-choice and choice tests in observation cages. Antennal rotation and ovipositor probing were found to be important in host searching and recognition, respectively, before a host was accepted for egg laying. Female moths oviposited significantly more eggs on larvae-infested cabbage than on intact uninfested cabbage and in particular more on leaves than on other parts of the cabbage plant. These results indicate the potential of developing a brassica host-derived kairomone attractant as a useful monitoring tool for female diamondback moths in area-wide integrated pest management programs that have a sterile insect technique (SIT) component.

In this study we identified a number of moth (Lepidoptera) species that are potential targets for the sterile insect technique (SIT), and we assessed the feasibility of using stable isotope signatures as markers to distinguish mass-reared from wild moth species. Large natural differences in the isotopic signatures of commercially available sugars render them novel markers for mass-reared insects. Sugar beet (Beta vulgaris L.; Caryophyllales: Amaranthaceae), a C₃ plant, has a stable isotopic signature (a measure of the ratio of the stable isotopes ¹³C:¹²C) of around -27‰ relative to Vienna Pee Dee Belemnite (VPDB; the international C isotope standard for the stable isotopes, ¹³C and ¹²C), and sugarcane (Saccharum spp.; Poales: Poaceae), a C₄ plant, has an isotopic signature of around -11‰. Thus by means of such a distinct isotope ratio in the sugar in the diet, mass-reared insects can be easily distinguished from wild insects with a high degree of certainty. It was shown that the method could be extended using a multiple isotope approach, with ¹⁵N or a full suite of C, N, S and O isotopes. Intrinsic isotope marking of mass-reared moths proved to be an accurate means of distinguishing wild from mass-reared populations, based on isotopic differences between the wild host plant species and the diets used in mass-rearing, which where possible, had been manipulated to contain the isotopically divergent sugar type. This intrinsic labeling using stable isotopes could be useful in the assessment of the quality of mass-reared moths, because a stable isotope is a marker that does not affect the insect in any detrimental manner.

Measurement of natural stable isotope signatures of insects has proven to be a useful method in determining their natal origin, feeding strategies and mating behavior, and has spawned a new discipline of insect isotope forensics. However the complexity of measuring isotope signatures using elemental analysis-isotope ratio mass-spectrometry (EA-IRMS) has encountered significant technical barriers, which together with costs, have hindered the advancement and wide spread adoption of this potentially useful approach. In the last decade simpler technologies to measure stable isotope ratios in samples based on laser spectroscopy have been developed. We foresee that these instruments will fuel the advancement and adoption of insect isotope forensics into pest management programs because they are easy to use, more affordable, and complement existing marking tools. However prior to the adoption of these new technologies it is necessary to test their fitness for purpose and explore their limitations. In this paper we set out to test the suitability of using combustion module-cavity ring-down spectroscopy (CM-CRDS) for measuring isotope ratios in an entomological context, covering marking of insects, mating behavior and dietary assessment. We carried out a series of experiments to compare the isotope measurements made with the CM-CRDS with the traditional method of EA-IRMS. Initially we compared measurements of carbon isotope values of a variety of common diet components used in insect rearing, which covered a wide range of carbon isotope values. There was good agreement in the isotope values obtained, using the CM-CRDS and the EA-IRMS with similar accuracy and precision. Secondly we compared the 2 methods to measure carbon isotope values of the common cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) samples, and again there was good agreement in the values obtained. Thirdly we tested whether the CM-CRDS could be used to determine the paternity of spermataphores, based on the differential isotope signatures of the paternal and maternal moths, as a result of differences in their rearing diets. The CM-CRDS proved to be fit for the purpose of measuring isotope values in the spermataphores of common cutworm and suggested that the CM-CRDS technology would be suitable for these isotope based mating studies in moths if the spermatophores were of sufficient mass. Finally we explored a number of the issues surrounding CM-CRDS and isotope technologies in general, e.g., logistical considerations, economics of operation, sample size, etc. We conclude that CM-CRDS is a suitable instrument for measuring stable isotope carbon signatures in moths, and most probably other sufficiently large insects and that CM-CRDS could be easily used in both operational and research contexts.

A simplified and improved rearing system was developed for the mahogany shoot borer, Hypsipyla grandella (Zeller) (Lepidoptera: Pyralidae) in the laboratory. Improvements were made in neonate rearing, larval containers, adult mating cages and the diet, making the rearing protocols more suitable for eventual large-scale rearing. The larval diet was modified by replacing ingredients with cheaper equivalents that also would be more readily available throughout the year. Cedar seeds in the larval diet were replaced with dried cedar leaves, which are available for longer periods during the year. There were no significant differences in pupal weight, duration of pupal development, fertility (egg hatch) and fecundity (number of eggs oviposited) between organisms reared on the new diet or the original diet. However, larval development took 2 d longer using the new diet. A new method for rearing neonate larvae is proposed, which includes the use of well plates that contain the modified diet. This allowed the rearing of larvae from 1st to 3rd instars without the need for fresh material. Neonate larvae reared on this diet were heavier and survived longer compared to larvae reared on fresh cedar leaves. Jars used for the larval rearing phase were replaced with Petri dishes. Mating and oviposition were accomplished inside the insectaries with air flow directed through the rearing cages. This is the first report of rearing H. grandella successfully under artificial conditions for 7 generations. The new rearing protocols will allow the maintenance of larger-sized colonies for further development of bio-rational pest management strategies that could include a sterile insect technique (SIT) component.

The sterile insect technique (SIT) requires the release of a large number of irradiated moths in the infested crop area targeted for suppression or eradication. Irradiated males must compete strongly with wild males for mating with conspecific wild females. Irradiated female moths are fully sterile and therefore when released do not pose a potential risk to the crop. However the close proximity of the released males and females may result in assortative mating among the irradiated moths, thereby undermining the competitive ability of the irradiated males. Furthermore, released females have partially depleted the reproductive potential of the colony, and will not contribute to further increases in the size of the colony in a mass-rearing facility. We tested 4 methods to separate European grapevine moth, Lobesia botrana (Dennis & Schiffermüller) (Lepidoptera: Tortricidae) males from females based on differences between males and females with respect to: (i) the number of abdominal segments of the pupae, (ii) the colors of wandering larvae, (iii) speed of maturation of larvae (protandry), and (iv) the lengths of the pupae. The sexing of 500 moths by the number of abdominal segments in the pupae was accomplished without any errors, but it was time consuming, tedious and required skill and experience. However, it should be possible to develop an automated apparatus to sort pupae by gender. Sexing by color differences did not provide clean separation because of large overlaps between the sexes in the green, blue and red spectra. Although male larvae entered the wandering phase significantly earlier than female larvae, in a large portion of the population the time of departure, some males and females departed simultaneously so that clean separation was possible for only about ¼ of the males or about ¼ of the females. A similar situation prevailed for emergence of adult males and females from pupae. On average, male pupae were significantly shorter than female pupae, and pupal length was found to be the most practical method for separating L. botrana males from females. For example, if all pupae < 5.4 mm were irradiated and released, they would include about 86% of all males and about 22% of all females—all of which would be the smallest and least fecund ones. This would allow about 78% of the females—including the largest and most fecund ones—to be retained for mass-rearing. At this time separation of the sexes based on pupal length is the most practical method.

Genetically modified maize contains an insecticidal gene from the soil bacterium Bacillus thuringiensis (Bt), which is an important component in integrated pest management strategies against lepidopteran pests of maize. A project is being implemented in the Western Cape of South Africa against the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), a pome fruit pest, using an area-wide integrated pest management approach with a sterile insect technique component. The project requires rearing of large numbers of the target pest for which an artificial diet that contains maize meal as the main ingredient is used. Most of the maize produced in South Africa is Bt maize, which is known to be toxic to codling moth. The aim of this study was to assess the impact of Bt maize in the diet of codling moth on its production parameters. Codling moths were reared for a period of 44 d on artificial diets that contained 5 different concentrations of Bt maize meal and a control using non-Bt maize. The use of Bt maize in the larval diet resulted in larval mortality, delayed larval development and larvae leaving the diet prematurely. Delayed larval development seemed to be the response with most negative consequences. Since optimal rearing of codling moth is not feasible using meal from genetically modified maize with insecticidal properties, another nutritious meal lacking an insecticidal component must be substituted in the artificial diet.

A bi-national program was established by Mexico and the United States to mitigate the threat of the cactus moth, Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae)—an invasive herbivore from South America—to native Opuntia spp. (Caryophyllales: Cactaceae) biodiversity and Opuntia-based industries. Mass-rearing, sterilization, and transport and release technologies assisted with the development of several control tactics including the sterile insect technique. Following the successful eradication of C. cactorum from Mexico and the elimination of C. cactorum from Alabama barrier islands, the bi-national program established an additional mass-rearing insectary for the production of sterile moths. Laboratory and field bioassays were conducted on sterile moths from both insectaries. Bioassays and assessments included moth mass, moth longevity, percentage of female moths mated at time of collection from the insectary, percentage of female moths mated 24 h after collection, flight ability, percentage recaptured after release in the field, and mean distance dispersed from release site. Data from the quality assessments and comparisons between the 2 insectaries were used as feedback mechanisms to make protocol changes in both rearing and handling that improved sterile moth quality and performance.

The sterile insect technique (SIT) is as an important component to the area-wide integrated management of the false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), which was successfully implemented in the Western Cape region of South Africa and subsequently expanded to citrus areas in the Eastern Cape region of South Africa. This integrated control program, which transports sterile moths from a rearing facility in Citrusdal, South Africa to orchards in both the Western and Eastern Cape, must continuously examine production, handling, processing, transport, and release protocols to ensure the delivery of high-quality sterile moths. While the use of cold temperature to immobilize moths is standard protocol for SIT programs to increase the density of moths for purposes of collecting, handling, irradiation, transport and release, some concern has been raised that rapid chilling and long cold temperature storage of moths may negatively impact field performance of some insectary-reared insects. We conducted trials to examine the effect of irradiation with 150 Gy of gamma rays and cold temperature storage on the performance of T. leucotreta moths released in citrus orchards. The radiation treatment did not significantly affect the performance of T. leucotreta moths released in citrus orchards. However, compared with moths held at room temperature, moths that were rapidly chilled were less likely to be captured in pheromone traps and less likely to disperse as great distances following release in citrus orchards. Additional research is needed to identify an alternative to rapid chilling and cold temperature storage that does not impair mating competitiveness and dispersal of irradiated T. Leucotreta adults. Procedural changes that will maintain or enhance sterile T. leucotreta moth quality and performance in the field, while allowing for the cost-effective handling and processing of the sterile moths, need to be considered.