Investigation of the spermathecal morphology, reproductive strategy and fate of stored spermatozoa in three important thysanopteran species.

In insects, females can keep sperm capable of fertilisation over a long period with the help of the spermatheca. The effectiveness of storing fertile sperm is expected to reflect in the reproductive strategy and, thus, the morphology of the involved organs. In this work, we focused on the relationship between reproduction and morphology in the haplodiploid Thysanoptera, especially if a loss of these traits occurs under thelytoky. The spermathecal morphology and the fate of stored spermatozoa were studied by microscopic techniques (high-resolution x-ray computed tomography and transmission electron microscopy) in three species with different reproductive modes and lifestyles (Suocerathrips linguis, Echinothrips americanus, Hercinothrips femoralis). Mating experiments were conducted to analyse the use of the transferred sperm in the thelytokous H. femoralis. Results show that the spermathecae are relatively simple, which can be explained by the availability of sperm and the short lifespan of the females. However, the spermatheca in H. femoralis seems to be vestigial compared to the arrhenotokous species and females do not use sperm for fertilisation. No substantial change was observed in the structure of spermatozoa, despite an enlargement of the sperm organelles being measured during storage in all three species. The results of this work demonstrate differences in the morphology of the spermatheca, especially concerning the reproduction mode, promoting the understanding of the complex interaction between morphology and behaviour.

Sperm ultrastructure in arrhenotokous and thelytokous Thysanoptera.

Thysanoptera are haplo-diploid insects that reproduce either via arrhenotoky or thelytoky. Beside genetically based thelytoky, this reproduction mode can also be endosymbiont induced. The recovery of these females from their infection again leads to the development of males. Functionality of these males ranges widely, and this might be associated with sperm structure. We analyzed the sperm ultrastructure in three different species belonging to both suborders with different reproduction systems via electron microscopy. Beside the different reproduction modes, and adaptations to their life style, the arrhenotokous species Suocerathrips linguis (Thysanoptera: Tubulifera) and Echinothrips americanus (Thysanoptera: Terebrantia) possess typical thysanopteran-like sperm structure. But endosymbiont-cured males from the thelytokous species Hercinothrips femoralis (Thysanoptera: Terebrantia) possess several malformed spermatozoa and a large amount of secretions in their testes. Spermiophagy seems to be typical. It indicates a highly conserved mechanism of the male developmental pathways, despite the observed decay. However, this decay would explain why in some species no stable arrhenotokous line can be re-established.

Description of the second instar larva of Thermothrips mohelensis Pelikán (Thysanoptera: Thripidae).

Thermothrips mohelensis Pelikán is an extremely rare thrips found in dry steppe grassland ecosystems (Pelikán 1949, 1995; Ulitzka 2019a). It has been reported from only a few locations and almost always in very low abundance: one female each has been collected in Germany (Ulitzka 2019a), Slovenia (Kucharczyk 2008) and Turkey (Tunç 1992), two females are known from Iran (Mirab-balou Chen 2013), three from Poland (Zawirska 1988; Kucharczyk 2007) and six from Western Russia (Schliephake 1977). Larger series including males and larvae are known only from Czechoslovakia (Pelikán 1949, 1995) and from Yakutia, East Siberia where-besides other specimens-the larvae discussed below have been collected (see Evdokarova Kucharczyk 2020). The fragmented and selective occurrence of this species in steppe habitats has been interpreted to represent scattered relict populations that may have remained from a more extensive distribution far back, possibly in the wide dry grasslands of the Late Pleistocene (Ulitzka 2019a). Similar to other specialist species of those barren habitats T. mohelensis is severely threatened by the general decline of these fragile ecosystems due to natural succession or anthropogenic impact (Kucharczyk 2008; Kucharczyk Kucharcyk 2008; Ulitzka 2019a). T. mohelensis is a thermophilous (zur Strassen 2003) and highly xerophilous species. This thrips was erroneously considered an hydrophilous wetland thrips (Bhatti 1998) due to a mistranslation (see Ulitzka 2019a). T. mohelensis lives and breeds monophagously in flowers of Galium (Rubiaceae), particularly G. verum but also G. mollugo (Pelikán 1949, 1995; Schliephake 1972; Tunç 1992).

Reproductive Behavior of Echinothrips americanus (Thysanoptera: Thripidae).

Most Thysanoptera possess a haplo-diploid reproductive mode and reproduce via arrhenotoky. Females can mature eggs successively throughout almost their entire life, but in most terebrantian thrips spermiogenesis is complete by adult male eclosion, and testes contain only mature spermatids. In parasitoid wasps this phenomenon of preadult spermiogenesis is described as prospermatogeny. It is unclear if prospermatogeny and this predetermined sperm quantity have implications for mating strategy and fitness. In this study, we give a detailed description of mating behavior of the thripine species Echinothrips americanus, which largely corresponds with the only available data of another species of this family, Frankliniella occidentalis (Thysanoptera: Thripidae). With investigations using light microscopy, we describe for the first time the chronological sequence of internal processes during copulation. The release of male accessory gland material followed subsequently by spermatozoa indicates production of a female-determined type 1 spermatophore. Despite prospermatogeny, males are able to inseminate 10 females with an equal amount of spermatozoa. It is only the quantity of glandular material that decreases with the number of previous copulations. Based on these new findings, the reproductive strategy of this species is discussed.

Male reproductive system and spermatogenesis of Limodromus assimilis (Paykull 1790).

Based on advanced light and electron microscopy, we describe the male reproductive system and sperm development of Limodromus assimilis. The genital tract consists of pairs of uni-follicular testes, spermatic ducts with diverticula regions, seminal vesicles, accessory glands, an unpaired ejaculatory duct and an aedeagus containing an internal sac equipped with sclerotic scales. Based on their morphology, we draw conclusions about their functions. After spermatogenesis within the follicle, the spermatozoa become released from the sperm cysts. The single spermatozoa move into the diverticula of the vasa deferentia I. Here, they become attached to central rods (spermatostyles), forming secondary conjugates (spermiozeugmata). The coordinated flagella movement of the conjugates possibly improves sperm velocity. Using super-resolution microscopy, we identified highly condensed reticulate chromatin in the lancet-shaped spermatozoa heads and the mitochondrial derivates of the flagella, likely formed by genomic and mitochondrial DNA, respectively. The results show, for the first time, sperm bundle formation in a Platynini species mainly corresponding to that found in Pterostichini species.

Fitness costs and life table parameters of highly insecticide-resistant strains of Plutella xylostella (L.) (Lepidoptera: Plutellidae) at different temperatures.

BACKGROUND: In many cases, resistance alleles have been associated with fitness costs and are often dependent on environmental factors such as temperature. Here, we studied the effects of temperature on the overall fitness, including development, survival and reproduction, of three insecticide-resistant and one susceptible strain of diamondback moth (DBM), Plutella xylostella (L.). RESULTS: The broader cross-resistance profile of the resistant strains previously selected by diamide and benzoylurea insecticides was tested. Cohort studies were conducted in the laboratory at three different temperatures (20 ± 1 °C, 25 ± 1 °C and 30 ± 1 °C), and involved fitness costs were estimated. We observed significant differences in the development time, with the susceptible strain showing a shorter developmental period from egg stage to adult stage compared with the resistant strains. Moreover, the resistant strains differed significantly between one another. Additionally, the population growth parameters varied among the strains, with the benzoylurea-resistant strain showing the highest costs affecting the overall fitness of this strain. A temperature of 30 °C was unfavourable for DBM development, resulting in a reduced fitness in all strains. CONCLUSION: Benzoylurea selection pressure on a diamide-resistant P. xylostella strain resulted in lowest reproduction parameters and the longest generation time, as well as doubling the time among all strains tested. This suggests significant effects on the overall fitness and population growth parameters for diamide-resistant populations pressured by benzoylureas under applied conditions. © 2017 Society of Chemical Industry.

Ryanodine receptor point mutations confer diamide insecticide resistance in tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae).

Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.

Male Pheromones Influence the Mating Behavior of Echinothrips americanus.

Two dibasic esters, the dimethyl ester of hexanedioic acid (dimethyl adipate, DBE-6) and the dimethyl ester of pentanedioic acid (dimethyl glutarate, DBE-5) were found in head-thorax extracts of male Echinothrips americanus. DBE-5 induced abdomen wagging and raising in males and females, which is typically exhibited when encountering a male. DBE-6 was avoided by males and was detected on mated, but not on virgin, females. Both substances applied to virgin females lead to females being ignored by males. The role of both substances is discussed with regard to the male mating system.

Active aggregation among sexes in bean flower thrips (Megalurothrips sjostedti) on cowpea (Vigna unguiculata).

Male sexual aggregations are a common territorial, mating-related or resource-based, behaviour observed in diverse organisms, including insects such as thrips. The influence of factors such as plant substrate, time of day, and geographic location on aggregation of thrips is uncertain, therefore we monitored the dispersion of male and female bean flower thrips (BFT), Megalurothrips sjostedti (Trybom) (Thysanoptera: Thripidae), on cowpea, Vigna unguiculata (L.) Walp. (Fabaceae), over three cowpea growth stages and across three cowpea-growing areas of Kenya. Our results indicated that for all the crop growth stages, the density of BFTs varied over the time of day, with higher densities at 10:00, 13:00, and 16:00 hours than at 07:00 hours. Thrips densities did not differ among blocks at the budding stage, but they did at peak flowering and podding stages. Dispersion indices suggested that both male and female BFTs were aggregated. Active male aggregation occurred only on green plant parts and it varied across blocks, crop stages, and locations. Similarly, active female aggregation was observed in peak flowering and podding stages. Such active aggregation indicates a semiochemical or behaviour-mediated aggregation. Identification of such a semiochemical may offer new opportunities for refining monitoring and management strategies for BFT on cowpea, the most important grain legume in sub-Saharan Africa.

Sternal gland structures in males of bean flower thrips, Megalurothrips sjostedti, and Poinsettia thrips, Echinothrips americanus, in comparison with those of western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae).

Sternal pores are important features for identification of male thrips, especially within the subfamily Thripinae. They vary in shape, size and distribution even between species of one genus. Their functional role is speculated to be that of sex- and/or aggregation pheromone production. Yet, sexual aggregations are not reported in Echinothrips americanus, known to have sternal pores, while we observed aggregations in Megalurothrips sjostedti, previously reported to lack them. We examined the sternal glands and pores of the thripine species E. americanus and M. sjostedti males, in comparison with those of Frankliniella occidentalis using light microscopy, as well as scanning and transmission electron microscopy. Pore plates of F. occidentalis were ellipsoid and medial on sternites III-VII, while in E. americanus they were distributed as multiple micro pore plates on sternites III-VIII. In M. sjostedti they appeared as an extremely small pore in front of the posterior margin of each of sternites IV-VII. Pore plate and pore plate area were distributed similarly on sternites III-VII in F. occidentalis. However, in E. americanus the total pore plate area increased significantly from sternites III to VIII. Ultrastructure of cells associated with sternal glands showed typical characteristics of gland cells that differ in size, shape and number. The function of sternal glands is further discussed on the basis of morphological comparisons with other thrips species.

Alkaloid metabolism in thrips-Papaveraceae interaction: recognition and mutual response.

Frankliniella occidentalis (Pergande), the Western Flower Thrips (WFT), is a polyphagous and highly adaptable insect of the order Thysanoptera. It has a broad host range but is rarely found on Papaveraceae, which might be due to deterrent effects of alkaloids present in most species of this family. In order to test the adaptive potential of WFT, we investigated its interaction with two Papaveraceae offered as sole feeding source. We found that WFT are able to live and feed on leaves of Eschscholzia californica and Chelidonium majus. Both plants respond to thrips feeding by the enhanced production of benzophenanthridine alkaloids. Furthermore, cell cultures of E. californica react to water insoluble compounds prepared from adult thrips with enhanced alkaloid production. During feeding, WFT take up benzophenanthridine alkaloids from either plant and from an artificial feeding medium and convert them to their less toxic dihydroderivatives. This was shown in detail with sanguinarine, the most cytotoxic benzophenanthridine. A similar conversion is used in plants to prevent self-intoxication by their own toxins. We conclude that WFT causes a phytoalexin-like response in Papaveraceae, but is able to adapt to such host plants by detoxification of toxic alkaloids.

An unexpected mixture of substances in the defensive secretions of the tubuliferan thrips, Callococcithrips fuscipennis (Moulton).

Adults and larvae of the thrips Callococcithrips fuscipennis (Moulton) (Thysanoptera: Tubulifera: Phlaeothripidae) live in the sticky wax masses of adult females of the felt scale insect Callococcus acaciae (Maskell) (Sternorrhyncha: Coccoidea: Eriococcidae). The scale is sessile and feeds on Kunzea shrubs (Myrtales: Myrtaceae). If stressed, the thrips produce droplets of secretions. The mixture contains pentadecane, tridecane, two monoterpenoids, hexadecyl butanoate, and smaller amounts of 15 other esters of long-chain unbranched alcohols identified as acetates, butanoates, hexanoates, and octanoates. The monoterpenoids are dolichodial, an iridoid, and an unknown substance with a mass spectrum very similar to that of anisomorphal and peruphasmal, diastereomers of dolichodial, but with a different retention time. Iridoids, butanoates, hexanoates, and octanoates have not been previously identified in Thysanoptera.

First detection of Wolbachia in arrhenotokous populations of thrips species (Thysanoptera: Thripidae and Phlaeothripidae) and its role in reproduction.

Insects in the order Thysanoptera are known for their haplodiploid reproduction, with most species reproducing by arrhenotoky. A few species, however, have only female populations and reproduce by thelytoky. Seven thrips species reproducing either by arrhenotoky [Frankliniella occidentalis (Pergande), Echinothrips americanus Morgan, Suocerathrips linguis Mound and Marullo, Gynaikothrips ficorum (Marchal)] or thelytoky [Hercinothrips femoralis (O.M. Reuter) and Parthenothrips dracaenae (Heeger)] or showing both reproduction modes (Thrips tabaci Lindeman) were screened for the presence of Wolbachia using 16S rDNA and ftsZ gene primers. The arrhenotokous reproducing species E. americanus, G. ficorum, and S. linguis tested positive for Wolbachia. The bacterium was not detected in F. occidentalis or T. tabaci; even the thelytokous population of T. tabaci was free of Wolbachia. Wolbachia was found in the thelytokous reproducing species H. femoralis and P. dracaenae. Antibiotic treatment of H. femoralis induced the production of males that copulated with females. The results confirmed Wolbachia to be present in thelytokous reproducing thrips species and proved the presence of the bacterium for the first time in some arrhenotokous reproducing thrips species.

A novel long-chained acetate in the defensive secretion of thrips.

Defensive secretions of adult and larval Suocerathrips linguis (Phlaeothripidae, Thysanoptera) were found to contain a long-chained acetate, (11Z)-11,19-eicosadienyl acetate, that was not previously known to occur naturally. This substance occurred together with octadecyl acetate and other long-chained acetates. The eicosadienyl acetate repels ants and spreads on the surface of such potential predators. The mixture can provide a long-lasting surface coating.

Tospovirus transmission depends on thrips ontogeny.

The acquisition of tospoviruses by thrips vectors is restricted to a well defined time period during the first and early second larval stages, when there is a temporary association between mid-gut, visceral muscles and salivary glands. This association is the result of a displacement of the brain into the prothoracic region by enlarged cibarial muscles. The subsequent loss of this association leads to a strong input of virus particles into the malpighian tubules via the haemocoel. Mechanical transmission through excrement and oviposition by adults is a possible alternative mode of virus transmission that requires investigation.