Variation in Thermal Sensitivity of Diapause Development among Individuals and over Time Predicts Life History Timing in a Univoltine Insect.

AbstractPhysiological time is important for understanding the development and seasonal timing of ectothermic animals but has largely been applied to developmental processes that occur during spring and summer, such as morphogenesis. There is a substantial knowledge gap in the relationship between temperature and development during winter, a season that is increasingly impacted by climate change. Most temperate insects overwinter in diapause, a developmental process with little obvious morphological change. We used principles from the physiological time literature to measure and model the thermal sensitivity of diapause development rate in the apple maggot fly Rhagoletis pomonella, a univoltine fly whose diapause duration varies substantially within and among populations. We show that diapause duration can be predicted by modeling a relationship between temperature and development rate that is shifted toward lower temperatures compared with typical models of morphogenic, nondiapause development. However, incorporating interindividual variation and ontogenetic variation in the temperature-to-development rate relationship was critical for accurately predicting fly emergence, as diapause development proceeded more quickly at high temperatures later in diapause. We conclude that the conceptual framework may be flexibly applied to other insects and discuss possible mechanisms of diapause timers and implications for phenology with warming winters.

Characterization of an agmatine N-acetyltransferase from Bactrocera dorsalis that modulates ovary development.

Agmatine N-acetyltransferase (AgmNAT), which catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine, is a member of the GCN5-related N-acetyltransferase family. So far, knowledge of the physiological roles of AgmNAT in insects is limited. Here, we identified one gene encoding protein homologous to that of Drosophila AgmNAT using sequence information from an activity-verified Drosophila AgmNAT in a BLAST search of the Bactrocera dorsalis genome. We expressed and purified B. dorsalis AgmNAT in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Our application of the screening strategy to BdorAgmNAT led to the identification of agmatine as the best amine substrate for this enzyme, with the highest kcat/Km value. We successfully obtained a BdorAgmNAT knockout strain based on a wild-type strain (WT) using the CRISPR/Cas9 technique. The ovary development of the BdorAgmNAT knockout mutants was delayed for 10 days compared with the WT specimens. Moreover, mutants had a much smaller mature ovary size and laid far fewer eggs than WT. Loss of function of BdorAgmNAT caused by RNAi with mature WT females did not affect their fecundity. These findings indicate that BdorAgmNAT is critical for oogenesis. Our data provide the first evidence for AgmNAT in regulating ovary development.

Seasonal fluctuation and alternative host plants of vegetable crop-infesting tephritids in non-vegetable growing areas in South Sudanese zone of Burkina Faso.

This study was carried out in 3 types of biotopes where vegetable crops are not grown to highlight their contribution to the dynamics of vegetable-infesting flies. To this end, a trapping system based on a sexual attractant, the Cuelure associated with an insecticide was set up in 18 biotopes (6 natural areas, 6 mango orchards, and 6 agroforestry parks) in the regions of Hauts Bassins and Cascades in the South-West of Burkina Faso. During the trapping monitoring, which was done every 2 wk to collect insects captured, fruits present in 3 types of biotopes were sampled and incubated for insect emergence. Ten Dacus (Fabricius) [Diptera: Tephritidae] species and Zeugodacus cucurbitae (Coquillett) [Diptera: Tephritidae] were trapped in the study area. The predominant species captured was Z. cucurbitae (52.93%) followed by Dacus punctatifrons (Karsch) [Diptera: Tephritidae] (29.89%) and Dacus humeralis (Bezzi) (12.71%). Six tephritid species were emerged from 6 wild fruit species belonging to Cucurbitaceae, Apocynaceae, and Passifloraceae families. Fruit flies were more abundant from Jul to Nov with peaks observed in Aug or Oct depending on the species. Citrullus colocynthis L. (Cucurbitaceae), Lagenaria sp. (Cucurbitaceae), Passiflora foetida L. (Passifloraceae), and Passiflora sp. acted as reservoir host plants of Dacus ciliatus (Loew), Dacus bivittatus (Bigot), Dacus vertebratus (Bezzi) [Diptera: Tephritidae], D. punctatifrons, and Z. cucurbitae, the major vegetable insect pests in West Africa. The 3 types of biotopes acted as suitable refuge areas of vegetable crop-infesting fruit flies either for the favorable microclimate or for the alternative host plants.

HSP gene superfamily in Aspongopus chinensis Dallas: unravelling identification, characterisation and expression patterns during diapause and non-diapause stages.

Aspongopus chinensis Dallas 1851, an insect of important economic value, faces challenges in artificial breeding due to mandatory diapause and limited access to wild resources. Heat shock proteins (Hsps) are thought to influence diapause in insects, but little is known about their role in A. chinensis during diapause. This study used genomic methods to identify 25 Hsp genes in A. chinensis, including two Hsp90, 14 Hsp70, four Hsp60 and five small Hsp genes, were located on seven chromosomes, respectively. The gene structures among the same families are relatively conserved. Meanwhile, the motif compositions and secondary structures of A. chinensis Hsps (AcHsps) were predicted. RNA-seq data and fluorescence quantitative PCR analysis showed that there were differences in the expression patterns of AcHsps in diapause and non-diapause stages, and AcHsp70-5 was significantly differentially expressed in both analysis, which was enriched in the pathway of response to hormone. All the results showed that Hsps play an important role in the diapause mechanism of A. chinensis. Our observations highlight the molecular evolution of the Hsp gene and their effect on diapause in A. chinensis.

Distribution analysis of TRH in Bactrocera dorsalis using a CRISPR/Cas9-mediated reporter knock-in strain.

Although the study of many genes and their protein products is limited by the availability of high-quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene-editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene-targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology-directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single-stranded oligodeoxynucleotide donor. This one-step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate-limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin-mediated behaviour of B. dorsalis.

Identification and functional verification of Y-chromosome-specific gene typo-gyf in Bactrocera dorsalis.

Genes on the Y chromosome play important roles in male sex determination and development. The identification of Y-chromosome-specific genes not only provides a theoretical basis for the study of male reproductive development, but also offers genetic control targets for agricultural pests. However, Y-chromosome genes are rarely characterized due to their high repeatability and high heterochromatinization, especially in the oriental fruit fly. In this study, 1 011 Y-chromosome-specific candidate sequences were screened from 2 to 4 h Bactrocera dorsalis embryo datasets with the chromosome quotient method, 6 of which were identified as Y-chromosome-specific sequences by polymerase chain reaction, including typo-gyf, a 19 126-bp DNA sequence containing a 575-amino acid open reading frame. Testicular deformation and a significant reduction in sperm number were observed after typo-gyf knockdown with RNA interference in embryos. After typo-gyf knockout with clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9 in the embryonic stage, the sex ratio of the emergent adults was unbalanced, with far more females than males. A genotype analysis of these females with the Y-chromosome gene MoY revealed no sex reversal. Typo-gyf knockout led to the death of XY individuals in the embryonic stage. We conclude that typo-gyf is an essential gene for male survival, and is also involved in testicular development and spermatogenesis. The identification of typo-gyf and its functional verification provide insight into the roles of Y-chromosome genes in male development.

Determination of temperature thresholds for the parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae), using life cycle simulation modeling: Implications for effective field releases in classical biological control of fruit flies.

The braconid parasitoid Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) is one of the most important natural enemies in classical biological control programs against tephritid fruit flies worldwide. In light of the spread of the invasive fruit fly species, Bactrocera dorsalis in Africa and beyond, there is a need to implement classical biological control. The current study aimed to determine temperature thresholds for D. longicaudata reared on B. dorsalis, using life cycle simulation modeling to guide informed parasitoid releases in Africa. Simulated parameters included thermal requirements, population growth parameters at different temperature requirements, suitable areas for the establishment, and the number of generations per year under projected climatic conditions. The lower thermal threshold for the development was estimated at 10.0°C, with a thermal constant (k) of 333.3-degree days, while the maximum temperature threshold was estimated at 33.69°C. Fecundity was highest at 25°C, with 177.3 eggs per female. Temperature significantly affected the population growth parameters of D. longicaudata, and the maximum value of the intrinsic rate of increase (rm) was 0.145 at 27°C. Results indicate that D. longicaudata could successfully establish in tropical and sub-tropical regions under current and future climatic conditions. However, a slight change in the suitable areas is expected by the year 2050 due to a slight and gradual rise in temperature. Our findings provide important information for further release of this parasitoid in Africa as well as designing pest management strategies to limit the spread and reduce the impact of fruit flies sustainably.

A polyphagous, tropical insect herbivore shows strong seasonality in age-structure and longevity independent of temperature and host availability.

Bactrocera tryoni is a polyphagous fruit fly that is predicated to have continuous breeding in tropical and subtropical Australia as temperature and hosts are not limiting. Nevertheless, in both rainforest and tropical agricultural systems, the fly shows a distinct seasonal phenology pattern with an autumn decline and a spring emergence. Temperature based population models have limited predictive capacity for this species and so the driver(s) for the observed phenology patterns are unknown. Using a demographic approach, we studied the age-structure of B. tryoni populations in subtropical Australia in an agricultural system, with a focus on times of the year when marked changes in population abundance occur. We found that the age-structure of the population varied with season: summer and autumn populations were composed of mixed-age flies, while late-winter and early-spring populations were composed of old to very old individuals. When held at a constant temperature, the longevity of adult reference cohorts (obtained from field infested fruits) also showed strong seasonality; the adults of spring and early autumn populations were short-lived, while late autumn and late winter adults were long-lived. While still expressing in modified landscapes, the data strongly suggests that B. tryoni has an endogenous mechanism which would have allowed it to cope with changes in the breeding resources available in its endemic monsoonal rainforest habitat, when fruits would have been abundant in the late spring and summer (wet season), and rare or absent during late autumn and winter (dry season).

Temperature-based phenology model to predict the development, survival, and reproduction of the oriental fruit fly Bactrocera dorsalis.

The oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae) is a major pest of fruit and vegetable production systems on several continents. The pest has invaded many countries, causing considerable impact on fruit production systems and commercialization. In this study we determined the relationship between temperature and development, survival and reproductive parameters of B. dorsalis on an artificial diet under laboratory conditions under 7 constant temperatures (10, 15, 20, 25, 30, 33 and 35 °C) with 70 ± 10% relative humidity and a photoperiod of L12:D12. We validated the laboratory results with a full life table analysis under semi-natural conditions in a screenhouse. We used the Insect Life Cycle Modeling (ILCYM) software for all mathematical models and simulations applied to all life history parameters. Bactrocera dorsalis completed its development at temperatures ranging between 15 and 33 °C with the mean developmental time of egg, larva, and pupa ranging between 1.46 and 4.31 days, 7.14-25.67 days, and 7.18-31.50 respectively. The models predicted temperatures ranging between 20 and 30 °C as favorable for development and survival, and 20 to 25 °C for optimal fecundity of B. dorsalis. Life table parameters showed the highest gross reproductive rate (GRR), net reproductive rate (Ro), intrinsic rate of increase (rm), and finite rate of increase (λ) between 25 and 31 ᵒC while generation time (T) and doubling time (Dt) were low at this interval. The effects of future climate change on B. dorsalis life history parameters were further investigated and the outcome from this study will help in the management of B. dorsalis in different agroecologies in the context of ongoing climate change.

Can Anastrepha fraterculus larval feeding influence chemotaxis and parasitism of Diachasmimorpha longicaudata and Aganaspis pelleranoi?

Anastrepha fraterculus (Diptera: Tephritidae) is a major barrier to fruit production and exportation. In Brazil, the native parasitoid Aganaspis pelleranoi (Hymenoptera: Figitidae) and the exotic parasitoid Diachasmimorpha longicaudata (Hymenoptera, Braconidae) stand out as biological control agents. Knowledge of the factors that affect interactions among parasitoids, A. fraterculus, and host fruits may enhance the use of these agents in biological control programmes. This study evaluated the chemotaxis and parasitism of A. pelleranoi and D. longicaudata females reared on A. fraterculus larvae and kept on an artificial diet, red guava (Psidium guajava) or apple (Malus domestica). Females of both parasitoid species that emerged from larvae raised on artificial diet, guava or apple, were tested to Y olfactometer choice tests. In the parasitism tests, both parasitoid species were made to choose between A. fraterculus larvae brushed with water, apple pulp or guava pulp. D. longicaudata females from artificial diet (control) did not distinguish between fruit odours; however, females of D. longicaudata from larvae kept in apple or guava directed to the odours of their original fruit. The greatest parasitism for D. longicaudata occurred in the units that contained the pulp in which the larvae grew. A. pelleranoi from artificial diet preferred guava odours, including the females kept in apple. Similar results were observed in the parasitism bioassays. Our results found that A. fraterculus larval feeding influenced search behaviour and parasitism of D. longicaudata, whereas A. pelleranoi rearing experience did not affect its host choices.

The abundance and diversity of fruit flies and their parasitoids change with elevation in guava orchards in a tropical Andean forest of Peru, independent of seasonality.

Lower elevations are generally thought to contain a greater abundance and diversity of insect communities and their natural enemies than higher elevations. It is less clear, however, how changes in seasons influence this pattern. We conducted a 2-year study (2013‒2014) in guava orchards located in a tropical Andean forest of Peru to investigate differences in fruit flies (Diptera: Tephritidae) and their parasitoid communities at two elevations and over two seasons. Fruit fly traps were installed, monitored, and guava fruits were sampled from eight orchards at low (800-950 m above sea level) and high (1,700-1,900 m above sea level) elevations and during the dry and rainy seasons. At each orchard, adult fruit fly trap captures and emergence of fruit flies and their parasitoids from guava fruit were quantified to determine their abundance and species composition. There was a greater abundance and species richness of fruit flies captured in traps at lower elevations, as well as higher abundance and species evenness of fruit flies that emerged from fruit, indicating that lower elevations are associated with larger fruit fly populations. The abundance, species richness and diversity of parasitoids were also greater at lower elevations. Consequently, guava fruit infestation and fruit fly parasitism rates were also greater at lower elevations. Seasonality also influenced fruit fly populations with a greater number of flies emerging from guava fruit and more fruit infested in the rainy season. However, seasonality had no effect on parasitoid population parameters or rate of parasitism, nor did it interact with elevation as an influence of populations of fruit flies or their parasitoids in guava orchards. This study highlights the importance of examining both elevation and seasonality for a better understanding of the population dynamics of fruit flies and their parasitoids in tropical agroecosystems.

Tephritid fruit flies have a large diversity of co-occurring RNA viruses.

Tephritid fruit flies are amongst the most devastating pests of horticulture, and Sterile Insect Technique (SIT) programs have been developed for their control. Their interactions with viruses are still mostly unexplored, yet, viruses may negatively affect tephritid health and performance in SIT programs, and, conversely, constitute potential biological control agents. Here we analysed ten transcriptome libraries obtained from laboratory populations of nine tephritid species from Australia (six species of Bactrocera, and Zeugodacus cucumis), Asia (Bactrocera dorsalis) and Europe (Ceratitis capitata). We detected new viral diversity, including near-complete (>99%) and partially complete (>80%) genomes of 34 putative viruses belonging to eight RNA virus families. On average, transcriptome libraries included 3.7 viruses, ranging from 0 (Z. cucumis) to 9 (B. dorsalis). Most viruses belonged to the Picornavirales, represented by fourteen Dicistroviridae (DV), nine Iflaviridae (IV) and two picorna-like viruses. Others were a virus from Rhabdoviridae (RV), one from Xinmoviridae (both Mononegavirales), several unclassified Negev- and toti-like viruses, and one from Metaviridae (Ortervirales). Using diagnostic PCR primers for four viruses found in the transcriptome of the Bactrocera tryoni strain bent wings (BtDV1, BtDV2, BtIV1, and BtRV1), we tested nine Australian laboratory populations of five species (B. tryoni, Bactrocera neohumeralis, Bactrocera jarvisi, Bactrocera cacuminata, C. capitata), and one field population each of B. tryoni, B. cacuminata and Dirioxa pornia. Viruses were present in most laboratory and field populations yet their incidence differed for each virus. Prevalence and co-occurrence of viruses in B. tryoni and B. cacuminata were higher in laboratory than field populations. This raises concerns about the potential accumulation of viruses and their potential health effects in laboratory and mass-rearing environments which might affect flies used in research and control programs such as SIT.

Comparative transcriptome analysis of different developmental stage of Bactrocera minax (Diptera: Tephritidae): Implication of the molecular basis of its obligatory diapause induction.

The Chinese citrus fruit fly, Bactrocera minax (Enderlein), a devastating pest in citrus, has an obligatory diapause at the pupal stage. Although the physiology of obligatory diapause has been well described, the molecular mechanisms underlying the process remain unknow. Here we investigated the molecular mechanisms of obligatory diapause induction in B. minax using high-throughput RNA-Seq data from second-instar larva (2L), third-instar larva (3L) and pupa (P) stages. A total of 116,402 unigenes were obtained, of which 54,781 unigenes were successfully annotated in public databases, and the differentially expressed genes in the 3L vs 2L, P vs 2L, and P vs 3L comparisons were identified. The cluster co-expression patterns of the differentially expressed genes revealed that significantly differentially-expressed genes in the pupal stage were predicted to be related to diapause induction. All differentially expressed genes were investigated by GO functional and KEGG pathway analysis, and the results showed that genes involved in processes such as 20-hydroxyecdysone (20E) biosynthesis, cell cycle and metabolic pathways are likely related to obligatory diapause induction in B. minax. These results provide important information on the transcriptome of the Chinese citrus fruit fly that can be used for further functional studies as well as contributing to our understanding of the molecular basis of obligatory diapause induction and suggesting potential molecular targets for the control of this pest.

Ovary-Specific Transcriptome and Essential Role of Nanos in Ovary Development in the Oriental Fruit Fly (Diptera: Tephritidae).

We used transcriptome analysis to research ovary development in Bactrocera dorsalis (Hendel). The ovary transcriptome of B. dorsalis yielded 66,463,710 clean reads that were assembled into 23,822 unigenes. After aligning to the Nr database in NCBI, 15,473 (64.95%) of the unigenes were matched to identified proteins. As determined by BLAST search, 11,043 (46.36%), 6,102 (25.61%), and 12,603 (52.90%) unigenes were each allocated to clusters via gene ontology, orthologous groups, and SwissProt, respectively. The Kyoto encyclopedia database of genes and genomes (KEGG) was further used to annotate these sequences, and 11,068 unigenes were mapped to 255 known pathways. Afterward, the genes that were possibly involved in oogenesis and ovary development were obtained from the transcriptome data and analyzed. Interestingly, seven ovary-specific genes were identified, including a Nanos gene that is involved in maintaining the primordial germ cells in many insects. Therefore, we further focused on the function of the BdNanos gene, and the gene was injected into B. dorsalis. As expected, the knocking down of Nanos gene expression led to significant inhibition of ovary development, suggesting an important role of this gene in the reproductive process of B. dorsalis. In summary, the present study provides an important reference for identifying the molecular mechanisms of oogenesis and ovary development in B. dorsalis. The BdNanos gene is crucial for ovary development in B. dorsalis and is therefore a potential new pest control target.

Knockdown of specific cuticular proteins analogous to peritrophin 3 genes disrupt larval and ovarian development in Bactrocera dorsalis (Diptera: Tephritidae).

Cuticular proteins (CPs) are critical components of the insect cuticle and play important roles in maintaining normal insect development and defense against various environmental stresses. The oriental fruit fly (Bactrocera dorsalis) is one of the most destructive pests worldwide, and its eight CPs analogous to peritrophin 3 (BdCPAP3) family genes have been identified in our previous study. In the present study, we further explored the possible roles of CPAP3 genes in B. dorsalis development. Each sequence of BdCPAP3 genes contained three conserved ChtBD2 (chitin-binding) domains. Spatial and temporal expression patterns revealed that the four BdCPAP3 genes (BdCPAP3-A1, B, E, and E2) might play important roles in larval pupariation of B. dorsalis. Moreover, treatment with a juvenile hormone analog (methoprene) significantly restricted expression of these four CPAP3 genes, whereas treatment with 20-hydroxy-ecdysone induced expression. The RNA interference (RNAi) results revealed that down-regulated CPAP3 genes led to significant delay of pupariation, and injection of dsBdCPAP3-E into 5-d-old B. dorsalis larvae caused approximately 40% mortality. Interestingly, we also confirmed that BdCPAP3-D2 was involved in B. dorsalis ovarian development. This study showed that some specific CPAP3 genes had crucial roles in B. dorsalis development, and these CP genes could be used as potential targets to control this pest via RNAi.

Big particles, best nutrition? Absorption and excretion of protein by Anastrepha obliqua larvae (Diptera: Tephritidae).

For purposes of mass-rearing fruit flies, nutrient intake through artificial diets is a challenge, artificial food content and processing should promote sufficient absorption and availability to ensure fly fitness. Bulking agents play an essential role in creating a quality diet, but its physical characteristics, such as particle size, may establish a better microenvironment for feeding and development. Currently, there is a lack of information about protein metabolism in mass-reared fruit flies. Therefore, we evaluated whether the particle size of the bulking agent affects the absorption and excretion of the proteins, as well as their effect on the life-history traits of Anastrepha obliqua. We determined the protein content of hemolymph and feces, as well as the presence of nitrogen end-products as indicators of their level of absorption in a diet elaborated with coarse and fine corn cob particles as a bulking agent. The bromatological composition showed that coarse particles increased the bioavailability and content of crude, digestible, and soluble protein for the diet and hemolymph protein of larvae alike. We found an inverse relationship between the protein content of the hemolymph and feces of the larvae. Ammonium was determined to be a product of the catabolism of proteins. Also, A. obliqua improved its development (yield and pupal weight) and fitness (adult emergence and flight ability) when larvae were reared on a coarse particle diet. In conclusion, a diet elaborated with a coarse bulking agent features increased protein bioavailability and nutritional quality, which, in turn, increases the life-history traits of A. obliqua.

Life history, dormancy regulation, reproductive physiology and basic behaviour of the subtropical fruit fly Rhagoletotrypeta pastranai (Diptera:Tephritidae).

With the purpose of broadening knowledge on the evolution of life history strategies and behaviour of fruit flies within the tribe Carpomyini, the natural history and mating behaviour of the poorly known species Rhagoletotrypeta pastranai Aczél, are described for the first time. Larvae of R. pastranai were recovered from infested Celtis tala Gillies ex Planch and Celtis iguanaea (Jacq.) Sarg. during a 2-month fruiting period. Adults emerged from the recovered pupae after an average of 144.9 ± 3.9 days for females and 143.2 ± 3.38 days for males, suggesting that most individuals became dormant. Results of a variable winter length study suggested that environmental factors other than winter length may regulate dormancy/diapause duration in this subtropical species. Under laboratory conditions, R. pastranai adults lived an average of 51.13 ± 3.06 days in case of females and 48.08 ± 3.76 days in case of males, and required 5-15 days to reach sexual maturity. Behavioural observations under confinement revealed scarce sexual activity but sufficed to determine that, as in other members of the tribe Carpomyini, R. pastranai exhibits a male resource defence mating system. We discuss our findings emphasizing the importance of documenting the natural history and behaviour of unknown species of family Tephritidae and additionally, we highlight the necessity of future research to understand factors regulating dormancy/diapause and the evolution of life history strategies and sexual behaviour of subtropical species.

Diversity of bacteria in different life stages and their impact on the development and reproduction of Zeugodacus tau (Diptera: Tephritidae).

Fruit flies usually harbor diverse communities of bacteria in their digestive systems, which are known to play a significant role in their fitness. However, little information is available on Zeugodacus tau, a polyphagous pest worldwide. This study reports the first extensive analysis of bacterial communities in different life stages and their effect on the development and reproduction of laboratory-reared Z. tau. Cultured bacteria were identified using the conventional method, and all bacteria were identified by high-throughput technologies (16S ribosomal RNA gene sequencing of V3-V4 region). A total of six bacterial phyla were identified in larvae, pupae, and male and female adult flies, which were distributed into 14 classes, 32 orders, 58 families and 96 genera. Proteobacteria was the most represented phylum in all the stages except larvae. Enterobacter, Klebsiella, Providencia, and Pseudomonas were identified by conventional and next-generation sequencing analysis in both male and female adult flies, and Enterobacter was found to be the main genus. After being fed with antibiotics from the first instar larvae, bacterial diversity changed markedly in the adult stage. Untreated flies laid eggs and needed 20 days before oviposition while the treated flies showed ovary development inhibited and were not able to lay eggs, probably due to the alteration of the microbiota. These findings provide the cornerstone for unexplored research on bacterial function in Z. tau, which will help to develop an environmentally friendly management technique for this kind of harmful insect.

On the complementarity of DNA barcoding and morphology to distinguish benign endemic insects from possible pests: the case of Dirioxa pornia and the tribe Acanthonevrini (Diptera: Tephritidae: Phytalmiinae) in Australia.

Fruit flies are considered economically important insects due to some species being agricultural pests. However, morphological identification of fruit fly adults and larvae can be difficult requiring a high level of taxonomic expertise, with misidentifications causing problematic false-positive/negative results. While destructive molecular techniques can assist with the identification process, these often cannot be applied where it is mandatory to retain a voucher reference specimen. In this work, we non-destructively (and partial-destructively) processed larvae and adults mostly belonging to the species Dirioxa pornia (Walker, 1849), of the poorly studied nonpest fruit fly tribe Acanthonevrini (Tephritidae) from Australia, to enable molecular identifications whilst retaining morphological vouchers. By retaining the morphological features of specimens, we confirmed useful characters for genus/species-level identification, contributing to improved accuracy for future diagnostics using both molecular and morphological approaches. We provide DNA barcode information for three species of Acanthonevrini known from Australia, which prior to our study was only available for a single species, D. pornia. Our specimen examinations provide new distribution records for three nonpest species: Acanthonevroides variegatus Permkam and Hancock, 1995 in South Australia, Acanthonevroides basalis (Walker, 1853) and D. pornia in Victoria, Australia; as well as new host plant records for D. pornia, from kangaroo apple, apricot and loquat.

Validation of degree-day models for predicting the emergence of two fruit flies (Diptera: Tephritidae) in northeast Egypt.

We estimated thermal developmental thresholds (T0 ) and degree-day (DD) constants for the immature stages of two tephritid pests, Bactrocera zonata (Saunders) and Ceratitis capitata (Weidenmann). Males of both species were trapped in an Egyptian guava orchard during the fruiting seasons of 2016 and 2017 and trap catches were compared with peak flights predicted by the DD model based on local weather data. Ceratitis capitata had faster development than B. zonata at 20 and 25 °C, but their overall developmental rate was similar at 30 and 35 °C. The thermal threshold of development (T0 ) of B. zonata was higher than that of C. capitata, indicating greater sensitivity to cold. Although 35 °C yielded the fastest development of both species, survival was higher at 30 °C, with B. zonata experiencing a slight advantage, suggesting better tropical adaptation. Immature development of B. zonata and C. capitata was estimated to require 338 and 373 d, respectively, and 616 and 424 DD for a complete generation. Trap catches over both seasons showed good correspondence to peaks of fly activity predicted by the DD models; deviations from expectation ranged from 0 to 7 d for both fly species. Both species had four overlapping generations per season, with B. zonata abundance peaking in the first generation in both years, but only in 2016 for C. capitata. The models predict about eight and 12 generations per year in northeast Egypt for B. zonata and C. capitata, respectively. These models should be useful for timing pest control measures to coincide with periods of peak fly activity in fruit orchards.