Insects In Diapause Research Articles

Schrödinger’s cat among insects: non-invasive Raman spectroscopy to determine live and dead insects in diapause

Schrödinger’s cat among insects: non-invasive Raman spectroscopy to determine live and dead insects in diapause

BmINR and BmAC6 genes involve in diapause regulation via the insulin/IGF signaling pathway in the silkworm (Bombyx mori)

Diapause of the silkworm (Bombyx mori) is an important ecological adaptation strategy regulated by multiple signaling pathways. As an evolutionarily conserved signaling pathway, the insulin/IGF signaling (IIS) pathway is essential in regulating lifespan, energy accumulation, and stress resistance in diapause insects. However, the regulatory mechanism of IIS on diapause in B. mori is still not fully understood. To investigate the role of the IIS pathway in regulating diapause, we first analyzed the transcription levels of the insulin receptor (BmINR) and its downstream gene adenylate cyclase 6 (BmAC6). The diapause-terminated eggs of a bivoltine strain QiuFeng (V2-QF) were incubated at 25 °C in natural room light for preparing diapause egg producers (DEPs) and at 17 °C in total darkness for preparing non-diapause egg producers (NDEPs), respectively. Then we investigated the effects of BmINR and BmAC6 on diapause phenotype and expression of diapause-related genes by RNA interference (RNAi) and overexpression techniques. The results showed that the mRNA expression levels of BmINR and BmAC6 in the head and ovary of NDEPs were higher than those in DEPs during the early and middle pupal stages. Furthermore, when BmINR was knocked down in the NDEPs, approximately 14.43% of eggs were in light red color and subsequently changed into gray-purple color after 48 h post-oviposition, then stayed in a diapause state. On the other hand, overexpression of BmINR or BmAC6 via recombinant baculoviruses did not cause any obvious phenotypic alterations in NDEPs, but it upregulated the expression of genes related to carbohydrate metabolism, which provides energy for embryonic growth and development. Therefore, it can be concluded that BmINR and BmAC6 genes regulate embryonic diapause in bivoltine B. mori.

Mmp-induced fat body cell dissociation promotes pupal development and moderately averts pupal diapause by activating lipid metabolism

In Lepidoptera and Diptera, the fat body dissociates into single cells in nondiapause pupae, but it does not dissociate in diapause pupae until diapause termination. Using the cotton bollworm, Helicoverpa armigera, as a model of pupal diapause insects, we illustrated the catalytic mechanism and physiological importance of fat body cell dissociation in regulating pupal development and diapause. In nondiapause pupae, cathepsin L (CatL) activates matrix metalloproteinases (Mmps) that degrade extracellular matrix proteins and cause fat body cell dissociation. Mmp-induced fat body cell dissociation activates lipid metabolism through transcriptional regulation, and the resulting energetic supplies increase brain metabolic activity (i.e., mitochondria respiration and insulin signaling) and thus promote pupal development. In diapause pupae, low activities of CatL and Mmps prevent fat body cell dissociation and lipid metabolism from occurring, maintaining pupal diapause. Importantly, as demonstrated by chemical inhibitor treatments and CRISPR-mediated gene knockouts, Mmp inhibition delayed pupal development and moderately increased the incidence of pupal diapause, while Mmp stimulation promoted pupal development and moderately averted pupal diapause. This study advances our recent understanding of fat body biology and insect diapause regulation.

The Fallacy of Year-Round Breeding in Polyphagous Tropical Fruit Flies (Diptera: Tephritidae): Evidence for a Seasonal Reproductive Arrestment in Bactrocera Species.

The genus Bactrocera (Diptera: Tephritidae) is endemic to the monsoonal rainforests of South-east Asia and the western Pacific where the larvae breed in ripe, fleshy fruits. While most Bactrocera remain rainforest restricted, species such as Bactrocera dorsalis, Bactrocera zonata and Bactrocera tryoni are internationally significant pests of horticulture, being both highly invasive and highly polyphagous. Almost universally in the literature it is assumed that Bactrocera breed continuously if temperature and hosts are not limiting. However, despite that, these flies show distinct seasonality. If discussed, seasonality is generally attributed to the fruiting of a particular breeding host (almost invariably mango or guava), but the question appears not to have been asked why flies do not breed at other times of the year despite other hosts being available. Focusing initially on B. tryoni, for which more literature is available, we demonstrate that the seasonality exhibited by that species is closely correlated with the seasons of its endemic rainforest environment as recognised by traditional Aboriginal owners. Evidence suggests the presence of a seasonal reproductive arrest which helps the fly survive the first two-thirds of the dry season, when ripe fruits are scarce, followed by a rapid increase in breeding at the end of the dry season as humidity and the availability of ripe fruit increases. This seasonal phenology continues to be expressed in human-modified landscapes and, while suppressed, it also partially expresses in long-term cultures. We subsequently demonstrate that B. dorsalis, across both its endemic and invasive ranges, shows a very similar seasonality although reversed in the northern hemisphere. While high variability in the timing of B. dorsalis population peaks is exhibited across sites, a four-month period when flies are rare in traps (Dec-Mar) is highly consistent, as is the fact that nearly all sites only have one, generally very sharp, population peak per year. While literature to support or deny a reproductive arrest in B. dorsalis is not available, available data is clear that continuous breeding does not occur in this species and that there are seasonal differences in reproductive investment. Throughout the paper we reinforce the point that our argument for a complex reproductive physiology in Bactrocera is based on inductive reasoning and requires specific, hypothesis-testing experiments to confirm or deny, but we do believe there is ample evidence to prioritise such research. If it is found that species in the genus undergo a true reproductive diapause then there are very significant implications for within-field management, market access, and biosecurity risk planning which are discussed. Arguably the most important of these is that insects in diapause have greater stress resistance and cold tolerance, which could explain how tropical Bactrocera species have managed to successfully invade cool temperate regions.

CYP314A1-dependent 20-hydroxyecdysone biosynthesis is involved in regulating the development of pupal diapause and energy metabolism in the Chinese citrus fruit fly, Bactrocera minax.

Diapause is an environmentally preprogrammed period of arrested development, and characterized by metabolic depression that can occur during any development stage of insect. The insect steroid hormone 20-hydroxyecdysone (20E), is converted from ecdysone by the cytochrome P450 enzyme shade (CYP314A1), and it exerts a potent effect on the induction and maintenance of diapause in obligatory diapause insects. However, the regulatory mechanism of 20E in obligatory diapause development remains unclear. In this study, the function of 20E in the pupal diapause of Bactrocera minax was investigated. We determined the expression pattern of Halloween P450 genes from larval to adult B.minax, and found differential expression of CYP314A1 from other P450 genes, with a high level in larvae and a low level in pupae. Dysfunction of CYP314A1 by dsCYP314A1 microinjection in third-instar larvae caused significant larval mortality or abnormal pupae. Compared with dsGFP and DEPC-water, dsCYP314A1-injected larvae had significantly reduced 20E titer and altered energy metabolism, and many individuals failed to pupate. Exogenous 20E microinjected into late third-instar larvae or 20E fed to early third-instar larvae both caused similar energy metabolism changes. The 20E-treated larvae of B.minax had reduced total lipids and increased amounts of trehalose and glycogen. Furthermore, 20E-treated diapause individuals showed rapid pupal development. The 20E biosynthesis was regulated by the expression of CYP314A1, and was involved in the induction and termination phase of obligate diapause by regulating energy metabolism in B.minax. © 2022 Society of Chemical Industry.

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

Abstract To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time‐consuming to maintain non‐diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15°C (15°C‐IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15°C‐IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15°C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non‐diapause eggs. By combining temperature and light controls, the improved 15°C‐IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15°C‐IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.

Regulation of gonad development and respiratory metabolism associated with food availability and reproductive diapause in the rice bug Leptocorisa chinensis

Food has an influence on many life history traits related to dormancy in insects. In our previous study with the rice bug Leptocorisa chinensis (Dallas) (Hemiptera: Alydidae), diapausing females transferred to conditions physically favorable for promoting the gonad development required food intake to resume gonad development, whereas males did not. These differences in response to food between males and females lead to two questions: (1) Was diapause in the starved females completed? (2) Were the starved males that resumed gonad development at the same physiological status as fed males? We tested these questions with two physiological indicators: gonad status and respiratory rate. Results indicate that starved females are able to complete diapause, but show depressed respiration relative to well-fed insects in diapause. Similar to females, starved males that resumed postdiapause gonad development also had depressed respiratory rate, and hence physiological status is presumed to be different between starved and fed individuals. In this study, it was also found that the photoperiodic signal is storable, whereas the food signal acts directly. The adaptive significance of regulation of gonad development and respiratory metabolism in relation to phenology of suitable host plant and reproductive strategy is discussed in both sexes.

Sexual differences in the crucial environmental factors for the timing of postdiapause development in the rice bug Leptocorisa chinensis

Although the regulation of reproductive diapause by environmental factors has been extensively studied in a variety of insect orders, sexual differences in the regulation of diapause are still poorly understood. We examined the effects of environmental factors including photoperiod, temperature and food on the regulation of reproductive diapause in both sexes of the rice bug Leptocorisa chinensis (Dallas) (Hemiptera: Alydidae) in the laboratory and under natural conditions. There was no significant difference in critical daylength (CDL) for diapause induction between the sexes. The CDL corresponded to the daylength in which development of the gonads was suppressed in half the insects caught in the field. Both sexes enter diapause at the same time in successive seasons. In diapause insects, on the other hand, there were sexual differences not only in retention of responsiveness to photoperiod and temperature but also in the food requirement, and these sexual differences lead to different timing of the resumption of reproductive development after overwintering.

Rates of metabolism in diapausing and reproductively active tropical butterflies, Euploea core and Euploea sylvester (Lepidoptera: Nymphalidae)

Abstract. Although the ecology of diapause has been widely studied in the field, the underlying physiological responses occurring in tropical diapausing insects remain virtually unexplored. This is especially the case with rates of respiration in diapausing tropical insect species. The present study compares rates of metabolism, as assessed by measurement of carbon dioxide production, between two species of diapausing and reproductively active tropical butterflies, Euploea core (W.S Macleay) and Euploea sylvester (Fabricius), independent of temperature. Measurement of metabolism over a day‐time/night‐time regime confirms that these tropical butterflies display a diurnal rhythm in accordance with many other tropical and temperate insect species, regardless of developmental state. In addition, diapausing Euploea butterflies display rates of carbon dioxide production only 28% lower than those of reproductively active butterflies, and can terminate diapause within days of receiving the correct cues. Maintaining a similar metabolic rate throughout diapause, as well as a rapid termination of diapause, would enable these tropical butterflies to respond immediately to larval host plant resources, without the disadvantage of missing optimum conditions, allowing the species to maximize their reproductive potential.

Expanding radiation quarantine treatments beyond fruit flies

Abstract1 The potential of ionizing radiation as a disinfestation treatment for insects other than tephritid fruit flies is discussed. Radiation quarantine treatments are unique in that insects are not killed immediately but rendered sterile or incapable of completing development.2 The most tolerant insect stage to radiation is that which is most developed. Female insects, but not always mites, are sterilized with equal or lower doses than males.3 Insects irradiated with sterilizing doses usually have shorter longevities than non‐irradiated ones. Low oxygen conditions often increase tolerance to radiation.4 Insects in diapause are not more tolerant of radiation than non‐diapausing ones.5 Some pests of several groups, such as aphids, whiteflies, weevils, scarab beetles, and fruit flies, may be controlled with doses ≤ 100 Gy. Some lepidopterous pests and most mites require about 300 Gy. Stored product moths may require as much as 1 kGy to sterilize, and nematodes could need > 4 kGy.6 Even though application of irradiation to pallet‐loads of produce could mean that up to three times the minimum required dose is applied to the perimeter of the pallet, many fresh commodities tolerate doses required for quarantine security against many quarantined pests. Irradiation is arguably the most widely applicable quarantine treatment from the standpoint of commodity quality.

The effects of 20‐hydroxyecdysone on breaking diapause ofDiprion piniL. (Hym., Diprionidae)

Abstract.The pine sawfly, Diprion pini L, like almost all Diprionidae, has complex diapause phenomena which affect the prepupal stages (eonymph and pronymph) in the cocoon. Experiments using 20‐hydroxyecdysone to break diapause of different populations of D. pini, were conducted. Doses of 2 and 10 μg/g of the hormone were injected into the insects in diapause, previously removed from their cocoons and placed in transparent capsules to observe their development. The experiments showed that 20‐hydroxyecdysone may cause D. pini to resume development and may even totally break the diapause. However, this reactivation seemed to require large doses of 20‐hydroxyecdysone. Moreover, the effects of applying this hormone varied according to the insect's origin, duration of the diapause maintenance and conditions of photoperiod and temperature present before and after the treatment. According to the cases, this resumption of development can affect a variable fraction of individuals and be more or less complete. It is suggested that the reaction to 20‐hydroxyecdysone in standard conditions could be used to measure the intensity of the diapause of natural D. pini populations.

DIAPAUSE INDUCTION IN THE HORN FLY (DIPTERA: MUSCIDAE)

AbstractLaboratory studies of diapause induction in the horn fly Haematobia irritans irritans (L.) indicated that the incidence of diapause was unrelated to daylength or the number of day–night cycles. Transfer experiments were conducted to estimate the beginning and end of diapause sensitivity and the required number of days for diapause induction. On average, sensitivity was between the ages 0.10 and 0.82 on a physiological age scale of immature development. The number of days required for 50% diapause was estimated as 19.1 days. A model was developed relating the proportion of insects in diapause to the number of days spent in the interval 0.10–0.82. Additional experiments revealed that diapause in the puparia was related to the mean temperatures experienced by immatures during the interval of sensitivity. An equation relating the proportion in diapause to temperature was also developed and compared with the required day number model. Both models provided similar estimates of the proportion of horn flies entering diapause.

REPRODUCTIVE BIOLOGY AND LABORATORY REARING OF CERANTHIA SAMARENSIS (VILLENEUVE) (DIPTERA: TACHINIDAE), A PARASITOID OF THE GYPSY MOTH, LYMANTRIA DISPAR (L.)

AbstractLaboratory observations on the biology of Ceranthia samarensis (Villeneuve), a tachinid parasitoid of the gypsy moth, were carried out in the laboratory at 22 °C day/15 °C night, 85–90% RH, and a 12L:12D photoperiod. Older (5–6 days post-eclosion) males mated readily with newly emerged females. Mating success was 60%. The gestation period of the mated females was 10–12 days. Laboratory-reared (on diet) second- and third-instar gypsy moth larvae feeding on newly grown foliage of Quercus rubra L. were exposed to gravid females of the parasitoid. Ceranthia samarensis ovolarviposited on the body of the host. The first-instar larva penetrated the host cuticle and developed internally, forming a respiratory funnel that caused a dark circular scar on the lateral side of the caterpillar. The average number of progeny (puparia) produced over the lifetime of a C. samarensis female was 55.0 ± 5.0 (SE) and average longevity was 4.1 ± 1.7 (SE) days. Diapause of the puparia was facultative and induced by temperatures below 20 °C combined with a 12L:12D photoperiod. In nondiapause individuals, total generation time was 22–40 days. To obtain diapause insects, puparia were stored for2monthsat 15 °C, 100% RH, and 12L:12D photoperiod for development of pharate adults. Cold storage at 2–4 °C and 100% RH for at least 5 months was required to obtain up to 75% eclosion after 5–9 days the following year.

Variations of biogenic amine levels in the brain of Pieris brassicae pupae during nondiapausing and diapausing development

AbstractThe post‐embryonic development of Pieris brassicae can either be continuous (under a long photoperiod) or interrupted at the pupal stage (induced by a short photoperiod); this phenomenon is termed facultative diapause. Several studies have indicated that certain brain mechanisms could be directly involved in the perception of variations in the photoperiod and could mediate some physiological effects particular to dormancy. Biogenic amines have been particularly implicated in the response to photoperiod variations and also in the regulation of development, especially in diapause induction and termination.High performance liquid chromatography with dual electrochemical detection has therefore been used to measure several biogenic amines in pupal nervous tissues at various stages of nondiapausing and diapausing development.During direct development, the levels of dopamine (DA) and N‐acetyldopamine (NADA: a DA metabolite) in brain were relatively high in 3‐day‐old pupae and at the end of pupal life (on the 8th day). Dihydroxyphenylacetic acid (another metabolite of DA) showed no variation. Serotonin was mainly observed in 2–3‐day‐old pupae but 5‐hydroxyindoleacetic acid was never detected. In young diapausing insects, similar variations of DA levels were observed even though a slight decrease of DA metabolites was noted. Serotonin appeared somewhat later (4–5 days) and attained higher levels. In late diapausing pupae, a marked increase in DA levels was observed, especially when pupae were kept at low temperature (4°C). During diapause, serotonin levels were reduced or even absent.

The role of juvenile hormone in the larval diapause of the European corn borer, Ostrinia nubilalis

The possible role of juvenile hormone (JH) in the induction and termination of larval diapause in the European corn borer, Ostrinia nubilalis, was investigated using topical applications of both JH I and a JH mimic as well as by monitoring JH titers with the Galleria bioassay. Neither JH nor the JH mimic ZR515 was capable of influencing diapause termination when administered topically. The Galleria bioassay revealed little or no JH in the hemolymph of mid diapause (>30 days) insects, indicating no demonstrable role for JH in diapause maintenance. When ZR515 was administered to nondiapause, newly ecdysed fifth instar larvae the pupal molting cycle was delayed. By use of photoperiodic regimes we were able to show that the molting delay was not equivalent to diapause induction. The Galleria bioassay showed differences in JH titer profiles between diapause and nondiapause animals during the final larval stadium. The nondiapause insects showed titers that decline rapidly to trace amounts following the molt to fifth instar then rose prior to pupation. The diapause insects had generally higher titers and exhibited a more gradual decline after the molt. No evidence was obtained to support the hypothesis that JH plays a key role in the induction, maintenance, or termination of larval diapause.

Toxicity of phosphine to the diapausing stages of Ephestia elutella, Plodia interpunctella and other lepidoptera

Diapausing larvae of the stored product moths Ephestia elutella (Hübner) and Plodia interpunctella (Hübner) were tolerant of phosphine only in short exposures, the tolerance of E. elutella being at least twice that of P. interpunctella. The tolerance of a freshly collected wild stock of each species was, at some temperatures, about two or three times that in a laboratory reared stock. The wild stock of E. elutella required concentration-time (CT) products of 36 mg hr/l in 4-day exposures at 10° and 15°C, and 18 mg hr/l in 2-day exposures at 20°C and above for 100% kill. The tolerance of E. elutella larvae in diapause was not dependent on the manner in which diapause was induced, maintained or subsequently terminated. Longer exposures to phosphine were more effective than shorter ones of similar CT product because an initial period of about a day in each exposure was required for insects in diapause to be killed. At 25°C, a concentration of 0.03 mg/l was below the threshold level for mortality of some larvae, long exposures at such very low concentrations proving ineffective. Diapausing pupae of an outdoor species, Pieris brassicae (L.), and eggs of the univoltine race of Bombyx mori (L.) were highly tolerant of phosphine at 10°C.

Dependence of RNA and protein synthesis on electrolyte concentrations

Insects in diapause are in a state where development and RNA and protein synthesis are arrested; chilling breaks the diapause and induces synthesis. Initiation of development may be followed by altered permeability, hence a role of electrolytes is envisaged. In fact, Na +, K +, and Mg 2+ induced RNA and protein synthesis greatly in wings and to a lesser extent in fat body in the diapaused pupae of the silkworm, Hyalophora cecropia. The electrolyte concentration for peak synthetic activity varied and was 120, 40, and 100 m M for NaCl, KCl, and MgCl 2 respectively. Osmotic pressure effects of these elctrolytes, employing freezing-point depression values, were compared with the effect of the non-electrolytic, sucrose. The results indicated that Na + or Mg 2+ but not K + may act via positive osmotic pressure.

Mutations et altérations dans la transmission des informations génétiques chez la drosophile par injection d'extraits de chrysalide diapausantes ( Pieris brassicae) traitées par des ptérines

The injection of a pterinic growth factor (folic acid) to insects in diapause under hormone-deficient conditions induced the formation of tumors. From these tumours, artificially induced in chrysalids of Pieris brassicae, a factor causing tumours was extracted which could be isolated by ultracentrifugation. The factor induced by the pterins (folic acid) is made up of light particles which contain DNA, and this DNA is the active constituent. It is a 4–8 S component after breakdown of the light particles. After injection of an extract of diapausal chrysalid treated with folic acid and a pterin mixture in Drosophila melanogaster, a reactivation of certain genes in the treated larvae-appearance of red pigment in the kynurenine cells of the alytic larval fat body inside the puparium, yellow pigment (sepiapterin) in the blood of larvae and pupae-and blockage of metamorphosis in permanent larvae and pupae were observed. Genetic modifications were evident in the surviving adults of D. melanogaster transmitted by the cytoplasm of females (there was a decrease in the number of males after the second generation). Injecting the surviving females with [ 3H]thymidine resulted in a considerable increase in DNA synthesis by the nurse cells and the migration of marked DNA from the nucleus to the cytoplasm. Administration to Drosophila larvae of an extract of diapausal chrysalids, treated with a mixture of hydrogenated pterin derivatives and various inhibitors in addition to folic acid, caused ebony mutants to appear, showing an abnormality in the melanogenic metabolism and mutants sepia and Bar in F 1 or F 2 offspring. The total absence of growth and possibly gonadotrophic hormones in Pieris chrysalids is absolutely necessary for producing mutations.

The function of fatty acids in the diapause of the khapra beetle Trogoderma granarium everts

1. 1. Dietary fatty acid fraction of larval faeces of Trogoderma granarium, containing palmitic, stearic, oleic and linoleic acids, induces and maintains the diapause in the larvae of the same species; linoleic acid is highly efficient either by feeding or by injection. 2. 2. A comparative examination of the fatty acid composition of diapause and nondiapause larvae of T. granarium showed that the diapause insects have an increased concentration of the bound myristoleic, palmitoleic and linoleic acids accompanied by a decrease in palmitic acid content. 3. 3. Mass spectra, infra-red and R f values for fatty acid esters of larval faeces are given.

Diapause Metabolism and Rearing Methods for the White-Lined Sphinx, Celerio lineata (Lepidoptera: Sphingidae)1

Recent findings indicate hormones from the braincorpora cardiaca complex may affect respiratory functions of cockroach fat body mitochondria (Keeley and Friedman 1969). A similar situation is seen in diapause which is both hormonally controlled and accompanied by prominent mitochondrial changes. The onset and termination of diapause in insects is regulated by neurosecretions from the brain (Williams 1952, DeWilde and DeBoer 1969), and during diapause, respiratory metabolism decreases to ⅙–1/20 of the normal rate (Schneiderman and Williams 1953, Adkisson et al. 1963, Stegwee 1964). This respiratory decrease is accompanied by both degeneration in mitochondrial ultrastructure and loss of enzymes, especially the cytochromes (Shappirio and Williams 1957, Stegwee 1964, Willis 1966). Because of these large responses, insects in diapause appear to be ideal subjects for studies on hormone-mitochondrial relationships.