Function and immuno-localization of aquaporins in the Antarctic midge Belgica antarctica

Aquaporin (AQP) water channel proteins play key roles in water movement across cell membranes. Extending previous reports of cryoprotective functions in insects, this study examines roles of AQPs in response to dehydration, rehydration, and freezing, and their distribution in specific tissues of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae). When AQPs were blocked using mercuric chloride, tissue dehydration tolerance increased in response to hypertonic challenge, and susceptibility to overhydration decreased in a hypotonic solution. Blocking AQPs decreased the ability of tissues from the midgut and Malpighian tubules to tolerate freezing, but only minimal changes were noted in cellular viability of the fat body. Immuno-localization revealed that a DRIP-like protein (a Drosophila aquaporin), AQP2- and AQP3 (aquaglyceroporin)-like proteins were present in most larval tissues. DRIP- and AQP2-like proteins were also present in the gut of adult midges, but AQP4-like protein was not detectable in any tissues we examined. Western blotting indicated that larval AQP2-like protein levels were increased in response to dehydration, rehydration and freezing, whereas, in adults DRIP-, AQP2-, and AQP3-like proteins were elevated by dehydration. These results imply a vital role for aquaporin/aquaglyceroporins in water relations and freezing tolerance in B. antarctica.     

Rapid cold-hardening in larvae of the Antarctic midge Belgica antarctica: cellular cold-sensing and a role for calcium

In many insects, the rapid cold-hardening (RCH) response significantly enhances cold tolerance in minutes to hours. Larvae of the Antarctic midge, Belgica antarctica, exhibit a novel form of RCH, by which they increase their freezing tolerance. In this study, we examined whether cold-sensing and RCH in B. antarctica occur in vitro and whether calcium is required to generate RCH. As demonstrated previously, 1 h at -5 degrees C significantly increased organismal freezing tolerance at both -15 degrees C and -20 degrees C. Likewise, RCH enhanced cell survival of fat body, Malpighian tubules, and midgut tissue of larvae frozen at -20 degrees C. Furthermore, isolated tissues retained the capacity for RCH in vitro, as demonstrated with both a dye exclusion assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based viability assay, thus indicating that cold-sensing and RCH in B. antarctica occur at the cellular level. Interestingly, there was no difference in survival between tissues that were supercooled at -5 degrees C and those frozen at -5 degrees C, suggesting that temperature mediates the RCH response independent of the freezing of body fluids. Finally, we demonstrated that calcium is required for RCH to occur. Removing calcium from the incubating solution slightly decreased cell survival after RCH treatments, while blocking calcium with the intracellular chelator BAPTA-AM significantly reduced survival in the RCH treatments. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) also significantly reduced cell survival in the RCH treatments, thus supporting a role for calcium in RCH. This is the first report implicating calcium as an important second messenger in the RCH response.     

Avoidance of intracellular freezing by the freezing-tolerant New Zealand Alpine weta Hemideina maori (Orthoptera: Stenopelmatidae)

Calorimetric analysis indicates that 82% of the body water of Hemideina maori is converted into ice at 10 degrees C. This is a high proportion and led us to investigate whether intracellular freezing occurs in H. maori tissue. Malpighian tubules and fat bodies were frozen in haemolymph on a microscope cold stage. No fat body cells, and 2% of Malpighian tubule cells froze during cooling to -8 degrees C. Unfrozen cells appeared shrunken after ice formed in the extracellular medium. There was no difference between the survival of control tissues and those frozen to -8 degrees C. At temperatures below -15 degrees C (lethal temperatures for weta), there was a decline in survival, which was strongly correlated with temperature, but no change in the appearance of tissue. It is concluded that intracellular freezing is avoided by Hemideina maori through osmotic dehydration and freeze concentration effects, but the reasons for low temperature mortality remain unclear. The freezing process in H. maori appears to rely on extracellular ice nucleation, possibly with the aid of an ice nucleating protein, to osmotically dehydrate the cells and avoid intracellular freezing. The lower lethal temperature of H. maori (-10 degrees C) is high compared to organisms that survive intracellular freezing. This suggests that the category of 'freezing tolerance' is an oversimplification, and that it may encompass at least two strategies: intracellular freezing tolerance and avoidance.     

Ultrastructural effects of lethal freezing on brain,muscle and Malpighian tubules from freeze-tolerant larvae of the gall fly,Eurosta solidaginis

In preparation for winter low temperatures, larvae of the gall fly, Eurosta solidaginis, accumulate the cryoprotectants glycerol, sorbitol, and trehalose. The fat body cells of these freeze-tolerant larvae can survive intracellular freezing to -80 degrees C for 48 h even though no whole larvae survive this treatment. We hypothesized that some other tissue was more susceptible to freezing and therefore may be responsible for larval death. This paper compares the ultrastructure of brain, muscle, and Malpighian tubules between non-lethally frozen and lethally frozen freeze-tolerant larvae. The nuclei of cortical brain cells from lethally frozen larvae exhibited clumped chromatin and nuclear membranes with occasional expansions or 'blebs' of the intermembranous space, while the cytoplasm contained swollen spheres of endoplasmic reticulum. In contrast, non-lethally frozen brain contained nuclei with evenly dispersed chromatin, smooth nuclear membranes and a cytoplasm free of swollen endoplasmic reticulum. Muscle tissue of lethally frozen larvae contained disrupted myofilaments surrounding the Z-line in comparison to non-lethally frozen muscle which had myofilaments extending all the way to the Z-line. Alterations of Malpighian tubule cells from lethally frozen larvae included an extracted cytoplasm with swollen and rounded mitochondria. In contrast, Malpighian tubule cells from non-lethally frozen larvae had a more concentrated cytoplasm with many rod-shaped mitochondria. Results show alterations to all three tissue types due to lethal freezing. The brain tissue contained the most observable alterations and therefore may be the most susceptible to lethal freeze damage.     

Evidence and bioassay for diuretic factors in the nervous system of larvalHeliothis virescens

Summary Diuretic factors were studied in the central nervous system of larvae of the tobacco budworm,Heliothis virescens, using [¹⁴C]urea as a sensitive indicator for water movement through isolated Malpighian tubules. The assay required Na⁺ and a pH of 6.0–6.2 for maximum activity. Malpighian tubules had high secretory activity in feeding larvae of the fifth instar, but the activity declined during the burrowing-digging stage that preceded pupation. Malpighian tubules from starved larvae showed a greater response to extracts of nervous tissues than did tubules from feeding larvae, and extracts showed a dose-response relationship with fluid secretion. Diuretic activity was distributed throughout all parts of the central nervous system with the brain having the most activity. Brain extracts increased fluid secretion by in vitro Malpighian tubules by more than 3-fold and doubled the rate of dye clearance from the hemolymph in vivo. Diuretic activity in nervous tissue extracts was unaffected by boiling but sensitive to proteases. Fluid secretion by in vitro tubules was increased by cAMP, dbcAMP, theophylline, octopamine and dopa. These studies provide evidence for the presence of diuretic factors in the central nervous system ofH. virescens larvae and describe a sensitive bioassay for these factors.Abbreviations AR activation ratio - cAMP cyclic AMP - dbcAMP dibutyryl cyclic AMP - dbcGMP dibutyryl cyclic GMP - Dopa dihydroxyphenylalanine - 5-HT 5-hydroxytryptamine - L1 larval instar - VCNS ventral central nervous system     

Survival at low temperature of larvae of the pine processionary moth Thaumetopoea pityocampa from an area of range expansion

1 Larvae of Thaumetopoea pityocampa (Lepidoptera: Notodontidae) develop throughout the winter, although their feeding activity and survival can be impaired by adverse climatic factors. The present study investigated the survival at low temperature of larvae originating from a population with range expansion in an alpine valley in Northern Italy.
2 The supercooling point of individually analysed larvae averaged at −7 °C. This value insufficiently described the cold hardiness of the larvae; 39% of the tested larvae were alive when returned to room temperature immediately after freezing. When larval colonies inside their nest were exposed to −17 °C for 1 h after gradual temperature decrease, survival was 70.4%.
3 Rearing of larvae in the laboratory at different day/night temperatures indicated an effect of cumulative chill injury on larvae. A logistic regression explained the relationship between negative thermal sum (h°C below 0 °C) received in the laboratory experiment and larval survival. A similar relationship was demonstrated between negative thermal sum and survival of larval colonies in the field.
4 In the laboratory experiment, some tested larvae were able to survive for up to 8 weeks without feeding depending on rearing temperature. As expected, feeding occurred only when larvae were reared at temperatures of 9 °C day/0 °C night.
5 We classify the larvae of T. pityocampa as being moderate freezing tolerant. The winter behaviour allows this species to track climate warming by a rapid expansion into those areas that become compatible with the insect's development.     

Differential localization and processing of apoptotic proteins in Malpighian tubules of Drosophila during metamorphosis

Drosophila development proceeds through three larval stages, before it pupates to reach adulthood. During pupation, larval tissues are destructed by programmed cell death and replaced by adult structures. Programmed cell death is a tightly regulated process accomplished by the induction of three closely linked pro-apoptotic genes reaper, hid and grim, ultimately leading to the activation of caspases, DRONC and DRICE and results in cell death. Unlike other larval tissues, Malpighian tubules are unique in not undergoing characteristic ecdysone-induced apoptosis and are carried to the adults. In this paper we show that apoptotic proteins, HID, GRIM, DRONC and DRICE are expressed in the Malpighian tubules, however they are sequestered in the nucleus. Significantly DRONC and DRICE are not enzymatically processed to active forms in the Malpighian tubules, however, ectopic expression of pro-apoptotic proteins leads to malformed Malpighian tubules and lethality. We also show that the Drosophila inhibitor of apoptotic protein 1, DIAP1, is localized and processed differently in Malpighian tubules. These results provide first evidence in favor of differential activity of apoptotic proteins in Malpighian tubules.     

Host‐mediated shift in the cold tolerance of an invasive insect

While many insects cannot survive the formation of ice within their bodies, a few species can. On the evolutionary continuum from freeze‐intolerant (i.e., freeze‐avoidant) to freeze‐tolerant insects, intermediates likely exist that can withstand some ice formation, but not enough to be considered fully freeze tolerant. Theory suggests that freeze tolerance should be favored over freeze avoidance among individuals that have low relative fitness before exposure to cold. For phytophagous insects, numerous studies have shown that host (or nutrition) can affect fitness and cold‐tolerance strategy, respectively, but no research has investigated whether changes in fitness caused by different hosts of polyphagous species could lead to systematic changes in cold‐tolerance strategy. We tested this relationship with the invasive, polyphagous moth, Epiphyas postvittana (Walker). Host affected components of fitness, such as larval survivorship rates, pupal mass, and immature developmental times. Host species also caused a dramatic change in survival of late‐instar larvae after the onset of freezing—from less than 8% to nearly 80%. The degree of survival after the onset of freezing was inversely correlated with components of fitness in the absence of cold exposure. Our research is the first empirical evidence of an evolutionary mechanism that may drive changes in cold‐tolerance strategies. Additionally, characterizing the effects of host plants on insect cold tolerance will enhance forecasts of invasive species dynamics, especially under climate change.     

Developmental study of thermotolerance and the heat shock response inLucilia cuprina (Weidemann)

The ability to withstand thermal stress in a laboratory population of the blowflyLucilia cuprina (measured as per cent adult survival following varying periods of exposure to elevated temperature up to a maximum of 48°C) was in the order pupa > larva > adult. Pre-exposure to a mild heat shock (37°C) induced tolerance to temperatures which were otherwise lethal. An analysis of heat shock-induced protein synthesis during development at similar elevated temperatures presented patterns corresponding to the above observations on thermotolerance. The induced level of synthesis of major heat shock proteins (viz., 79, 69, 28, 20 and 19 kDa) were greater in larval tissues than in most of the adult tissues except gonads. The response varied between young (2 days) and old (30 days) adults in a tissue-specific manner. In general, heat shock protein 69 kDa was most abundant in all the tissues studied. Control as well as heat shocked Malpighian tubules of adults uniquely showed two major [³⁵S]methionine labelled bands corresponding to approximately 62 and 64 kDa. Immunoblots showed the 62 kDa protein to cross react with an antibody againstHelioihis HSP60. Although the synthesis of the 62 kDa polypeptide was prominent only in Malpighian tubules of adult blowflies, nearly equal levels of this HSP60 family polypeptide were present in all tissues (control as well heat shocked) except the larval salivary glands.     

Geographical and diapause-related cold tolerance in the blow fly,Calliphora vicina

Three geographical strains of the blow fly, Calliphora vicina, were tested for cold tolerance at 0 degrees, -4 degrees and -8 degrees C. Survival to eclosion after 1 to 18 days of cold exposure was greater for diapause-destined larvae than for nondiapause-destined larvae of the two northern strains (Nallikari, Finland 65 degrees N and Edinburgh, Scotland 55 degrees N) but not for the southernmost strain (Barga, Italy 44 degrees N) where no clear differences were apparent. Diapause-destined larvae of the Edinburgh strain were more cold tolerant than those from Nallikari, at both -4 degrees and -8 degrees C, a difference possibly attributable to the long-lasting snow cover in the more northern locality, which might insulate the overwintering soil microclimate. At 0 degrees C, however, Nallikari larvae were more cold tolerant than Edinburgh or Barga. This was also the case for nondiapause-destined larvae, indicating that cold tolerance may occur, in part, independently of the diapause programme. In all three strains diapausing larvae were more cold tolerant than same-age (nondiapausing) pupae. For Nallikari, but not Barga, wandering larvae from short-day exposed flies, therefore initially programmed for diapause, but diverted from the diapause pathway by larval breeding at 19 degrees C, were significantly more cold tolerant than nondiapause larvae from long-day parents, indicating some maternal regulation of larval cold tolerance. There was, however, no evidence for an additional cold hardiness in larvae acclimatised to cold by a gradual reduction of temperature.     

Cloning and expression analysis of midgut chymotrypsin-like proteinases in the tobacco hornworm

Digestion of proteins in the midgut of lepidopteran larvae relies on different trypsin and chymotrypsin isoforms. In this study we describe three chymotrypsin-like proteinases (CTLP2-4) from the larval midgut of Manduca sexta, which are closely related to CTLP1 and less closely related to another chymotrypsin (CT), two previously described proteinases present in the larval midgut of M. sexta. CTLP1-4 fit perfectly into a novel subgroup of insect CTLPs by sequence similarity and by the replacement of GP by SA in the highly conserved GDSGGP motif. When we examined CTLP expression in different tissues, most of the proteinases were predominantly expressed in the anterior and median midgut, while some were found in the Malpighian tubules. When we examined CTLP expression at different physiological states, we observed that the CTLP mRNA amounts did not differ considerably in feeding and starving larvae except for CTLP2, whose mRNA dropped significantly upon starvation. During moulting, however, the mRNA amounts of all CTLPs dropped significantly. When we immunologically examined CTLP amounts, mature proteinases were only detectable in the gut lumen of feeding and re-fed larvae, but not in that of starving or moulting larvae, suggesting that CTLP secretion is suspended during starvation or moult.     

MOLECULAR CHARACTERIZATIONS OF TWO CYTOCHROME P450 GENES ENCODING CYP6A41 AND CYP6EK1 FROM THE ORIENTAL FRUIT FLY,Bactrocera dorsalis (DIPTERA: TEPHRITIDAE)

Two P450 genes encoding CYP6A41 and CYP6EK1 were cloned from the oriental fruit fly using polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. CYP6A41 and CYP6EK1 contained open reading frames of 1,530 and 1,524 nucleotides that encode 510 and 508 amino acid residues, respectively. The putative proteins shared 44% identity with each other. Phylogenetic analysis showed that CYP6A41 and CYP6EK1 were most closely related to Ceratitis capitata CYP6A10 and CYP6A subfamily. Expression patterns of the two genes in different geographical populations (Yunnan, Hainan, Dongguang, and Guangzhou), developmental stages (eggs, larvae, pupae, and adults), and tissues (midguts, fat bodies, and Malpighian tubules) were analyzed by real‐time quantitative PCR (RT‐qPCR) methods. The results showed that the expression levels of CYP6EK1 were significantly different among the four populations, but were not different for CYP6A41. Both the expressions of CYP6A41 and CYP6EK1 were development specific and had significantly higher levels in the larval stage. The expression of CYP6A41 did not vary among the midgut, fat body, or Malpighian tubules; however, CYP6EK1 expression was higher in the Malpighian tubules. The results suggest that CYP6A41 and CYP6EK1 might be involved in detoxification of xenobiotic compounds that were harmful to larval flies or development. Moreover, high expression of CYP6EK1 in the Malpighian tubules also implied participation in detoxification.     

Comparative histopathology of infection byOvavesicula popilliae [Microsporida: Pleistophoridae] in larval and adult Japanese beetles,Popillia japonica

The host response to infection and tissue susceptibility of larval and adult Japanese beetles,Popillia japonica Newman, to the microsporidium,Ovavesicula popilliae Andreadis & Hanula, are reported. The normally transparent Malpighian tubules of Japanese beetle larvae, were hypertrophied and white in color when infected withO. popilliae, a microsporidian which also infects larval fat body, epidermis and pericardial cells. In addition to these tissues, œnocytes and tracheal epithelial cells were also infected in adults. Adult and larval reactions to infection included hypertrophied cells and melanization of the pericardium, but only larvae exhibited an intense inflammatory response. The discoloration of the pericardium most likely resulted from an accumulation of melanin.      

Photoperiodic and thermal regulation of development and cold hardiness in larvae of the clover leaf weevil, Hypera punctata

Effects of photoperiod and temperature on the development and cold hardiness were investigated in larvae of Hypera punctata. At a relatively low temperature (15 degrees C), the larvae fed less and developed more slowly under a 12L:12D (SD) photoperiod than under a 16L:8D photoperiod (LD). SD larvae had lower gut weight against the whole body weight and lower supercooling point (SCP) than the LD counterparts for the same instar and same body weight. This was because the larval SCP is markedly affected by the quantity of the gut content. Laboratory experiments indicated that the low temperature mortality of this larvae occurred mainly due to freezing irrespective of the photoperiod and temperature, suggesting that the lower lethal temperature (LLT) depends on the supercooling ability of larvae. The SD larvae tended to have a lower SCP and hence a lower LLT than the LD counterparts at 15 or 10 degrees C, unlike at 20 degrees C. Thus, the slower larval development under SD conditions at relatively low temperatures may prevent larvae from reaching the later instar, which have a higher SCP and thus less cold tolerance, during the coldest season. The suppressed feeding activity under SD conditions would lower the SCP, thereby reducing the possibility of lethal tissue freezing. Such a photoperiodic and thermal regulation of the larval development and the supercooling ability appear to represent adaptive mechanisms for winter survival in this beetle.     

Absorption of toxic beta-glucosides produced by plants and their effect on tissue trehalases from insects

Trehalases present in body wall, Malpighian tubules, fat body, midgut and haemolymph from Tenebrio molitor (Coleoptera), Musca domestica (Diptera), Spodoptera frugiperda and Diatraea saccharalis (Lepidoptera) were assayed in the presence and absence of toxic beta-glucosides produced by plants or their aglycones. The glucosides used were phlorizin, amygdalin, prunasin and the aglycone mandelonitrile. In addition, T. molitor and S. frugiperda trehalases were assayed with and without esculin. More than 60% of total trehalase activity was found in the midgut of these insects. As a rule, trehalases present in each insect were inhibited by at least two of the glucosides. Prunasin was the best inhibitor in tissues with highest trehalase activity. S. frugiperda beta-glucosidases were not able to hydrolyze esculin. Nevertheless, their larval midguts absorb the intact glucoside that is recovered from the fat body, Malpighian tubules and mainly from haemolymph. Mature larvae fed on a diet containing 3 mM (0.1%) esculin have 0.2 mM esculin in their haemolymph, and weigh 60% of control larvae. In vitro, haemolymph trehalase activity is abolished by 0.5 mM esculin. This inhibition may play a role in the decrease of body weight and in animal survival. S. frugiperda larvae reared in 0.1% amygdalin-containing diet present higher trehalase activity in tissues than the larvae reared in 0.1% esculin-containing diet. Higher trehalase activity should be the reason why the S. frugiperda development is not impaired by 1% dietary amygdalin, in contrast to what is observed when insects are reared in 0.1% esculin. The data suggest that many plant beta-glucosides are toxic because they inhibit trehalase, a key enzyme controlling glucose availability in insects.     

The effect of infection with Dirofilaria immitis (dog heartworm) on fluid secretion rates in the Malpighian tubules of the mosquitoes Aedes taeniorhynchus and Anopheles quadrimaculatus

Dirofilaria immitis, the parasitic nematode causing the disease dog heartworm, is transmitted by female mosquitoes. During their development, larval nematodes reside in the cells of the Malpighian tubules of these mosquitoes for approx 13 days. We have examined the effect of the presence of these large intracellular parasites on the main physiological function of the Malpighian tubules, i.e. fluid secretion. Rates of fluid secretion were examined in vitro using both normal and infected tubules of the mosquito species Aedes taeniorhynchus and Anopheles quadrimaculatus. Tubules of A. quadrimaculatus show changes in trasport rate during the reproductive cycle. Those of A. taeniorhynchus do not. Infection with larvae of D. immitis had no effect on the rate of fluid secretion in tubules of A. quadrimaculatus. In A. taeniorhynchus by contrast, the tubules show decline in transport with time following infection. The reduction in transport capacity is proportional to the number of worms infecting the tubule. The present paper and separate ultrastructural studies demonstrate that parallel changes in microvillar ultrastructure and epithelial transport rates occur in response to infection by the parasite. In both species examined, the survival of the mosquitoes and their vector potential are determined by factors other than the transport capacities of the infected Malpighian tubules.     

A technique for preparing polytene chromosomes from Aedes aegypti (Diptera,Culicinae)

Polytene chromosome preparations were obtained from larval, pupal and adult female Malpighian tubules of Aedes aegypti. The Malpighian tubules of the pupae (0-4 h old) from larvae reared at 20 C provided the best cytogenetic analysis. The interaction of nucleic acids and proteins that influence the spreading of the chromosomes could be reduced with the preparation technique of the sheets submitted to a stronger treatment starting with the hypotony of tissue and successive bathings with acetic acid. A simple technique should facilitate molecular cytogenetics used in the location of resistance and vector competence genes.     

Ultrastructural changes in the muscles,midgut, hemopoietic organ,imaginal discs and Malpighian tubules of the mosquito Aedes aegypti larvae infected by the fungus Coelomomyces stegomyiae

Fungi belonging to the genus Coelomomyces can infect mosquito larvae and develop within the larval hemocoel. To examine fungal development, Aedesaegypti larvae infected with Coelomomyces stegomyiae Keilin were fixed, embedded and sectioned for both light and electron microscopy. While fungal hyphae of C. stegomyiae did not invade cells other than the cuticular epithelial cells, they did penetrate a number of tissues including muscles, midgut, hemopoietic organ, imaginal discs, and Malpighian tubules. This revised version was published online in July 2006 with corrections to the Cover Date.