3rd Instar Stage Research Articles

Gut fungal diversity across different life stages of the onion fly Delia antiqua

A significant number of microorganisms inhabit the intestinal tract or the body surface of insects. While the majority of research on insect microbiome interaction has mainly focused on bacteria, of late multiple studies have been acknowledging the importance of fungi and have started reporting the fungal communities as well. In this study, high-throughput sequencing was used to compare the diversity of intestinal fungi in Delia antiqua (Diptera: Anthomyiidae) at different growth stages, and effect of differential fungi between adjacent life stages on the growth and development of D. antiqua was investigated. The results showed that there were significant differences in the α and β diversity of gut fungal communities between two adjacent growth stages. Among the dominant fungi, genera Penicillium and Meyerozyma and family Cordycipitaceae had higher abundances. Cordycipitaceae was mainly enriched in the pupal and adult (male and female) stages, Penicillium was mainly enriched in the pupal, 2nd instar and 3rd instar larval stages, and Meyerozyma was enriched in the pupal stage. Only three fungal species were found to differ between two adjacent growth stages. These three fungal species including Fusarium oxysporum, Meyerozyma guilliermondii and Penicillium roqueforti generally inhibited the growth and development of D. antiqua, with only P. roqueforti promoting the growth and development of female insects. This study will provide theoretical support for the search for new pathogenic microorganisms for other fly pests control and the development of new biological control strategies for fly pests.Graphical Three fungal species were found to differ between two adjacent growth stages. These three fungal species including Fusarium oxysporum, Meyerozyma guilliermondii and Penicillium roqueforti generally inhibited the growth and development of D. antiqua, with only P. roqueforti promoting the growth and development of female insects.

Energetic demands regulate sleep-wake rhythm circuit development

Sleep and feeding patterns lack strong daily rhythms during early life. As diurnal animals mature, feeding is consolidated to the day and sleep to the night. In Drosophila, circadian sleep patterns are initiated with formation of a circuit connecting the central clock to arousal output neurons; emergence of circadian sleep also enables long-term memory (LTM). However, the cues that trigger the development of this clock-arousal circuit are unknown. Here, we identify a role for nutritional status in driving sleep-wake rhythm development in Drosophila larvae. We find that in the 2nd instar larval period (L2), sleep and feeding are spread across the day; these behaviors become organized into daily patterns by the 3rd instar larval stage (L3). Forcing mature (L3) animals to adopt immature (L2) feeding strategies disrupts sleep-wake rhythms and the ability to exhibit LTM. In addition, the development of the clock (DN1a)-arousal (Dh44) circuit itself is influenced by the larval nutritional environment. Finally, we demonstrate that larval arousal Dh44 neurons act through glucose metabolic genes to drive onset of daily sleep-wake rhythms. Together, our data suggest that changes to energetic demands in developing organisms trigger the formation of sleep-circadian circuits and behaviors.

Energetic demands regulate sleep-wake rhythm circuit development.

Sleep and feeding patterns lack strong daily rhythms during early life. As diurnal animals mature, feeding is consolidated to the day and sleep to the night. In Drosophila, circadian sleep patterns are initiated with formation of a circuit connecting the central clock to arousal output neurons; emergence of circadian sleep also enables long-term memory (LTM). However, the cues that trigger the development of this clock-arousal circuit are unknown. Here, we identify a role for nutritional status in driving sleep-wake rhythm development in Drosophila larvae. We find that in the 2nd instar larval period (L2), sleep and feeding are spread across the day; these behaviors become organized into daily patterns by the 3rd instar larval stage (L3). Forcing mature (L3) animals to adopt immature (L2) feeding strategies disrupts sleep-wake rhythms and the ability to exhibit LTM. In addition, the development of the clock (DN1a)-arousal (Dh44) circuit itself is influenced by the larval nutritional environment. Finally, we demonstrate that larval arousal Dh44 neurons act through glucose metabolic genes to drive onset of daily sleep-wake rhythms. Together, our data suggest that changes to energetic demands in developing organisms trigger the formation of sleep-circadian circuits and behaviors.

Feeding assimilation of fall armyworm (Spodoptera frugiperda J.E Smith) (Lepidoptera: Noctuidae) larvae on several host types

Spodoptera frugiperda (Noctuidae: Lepidoptera) is a pest that is easy to adapt and quickly develops. S. frugiperda can switch to crops other than maize as the primary host. Food assimilation must be tested to determine which plants S . frugiperda larvae prefer. This research was conducted to determine the feeding assimilation of S. frugiperda larvae and determine plants that can be alternative hostsinstead of maize . This study was arranged using a completely randomized design (CRD) with three treatments and five replications to obtain 15 experimental units. Larvae feed consists of rice, maize, and Napier grass. Feed is given during their 3rd instar stage until it becomes a prepupa. The suitability of plants as a food source for insects can be determined by a food assimilation test calculated using the Gravimetric method. The findings from the observations of food assimilation of S. frugiperda larvae in larvae-fed maize had higher consumption rate, growth rate, and digestibility than larvae-fed Napier grass. The conversion efficiency of digested food in grass-consuming larvae is 24.173%, higher than maize, 10.227%, and rice 0.00%. In compliance, the efficiency of food utilization was higher for larvae-fed Napier grass compared to those fed maize and rice.

Chronic exposure to 2,2′-azobis-2-amidinopropane that induces intestinal damage and oxidative stress in larvae of Drosophila melanogaster

Embryonic development is exceptionally susceptible to pathogenic, chemistry and mechanical stressors as they can disrupt homeostasis, causing damage and impacted viability. Oxidative stress has the capacity to induce alterations and reshape the environment. However, the specific impacts of these oxidative stress-induced damages in the gastrointestinal tract of Drosophila melanogaster larvae have been minimally explored. This study used 2,2-azobis (2-amidinopropane) dihydrochloride (AAPH), a free radical generator, to investigate oxidative stress effects on Drosophila embryo development. The results showed that exposing Drosophila eggs to 30 mM AAPH during 1st instar larva, 2nd instar larva and 3rd instar larva stages significantly reduced hatching rates and pupal generation. It increased the activity of antioxidant enzymes and increased oxidative damage to proteins and MDA content, indicating severe oxidative stress. Morphological changes in 3rd individuals included decreased brush borders in enterocytes and reduced lipid vacuoles in trophocytes, essential fat bodies for insect metabolism. Immunostaining revealed elevated cleaved caspase 3, an apoptosis marker. This evidence validates the impact of oxidative stress toxicity and cell apoptosis following exposure, offering insights into comprehending the chemically induced effects of oxidative stress by AAPH on animal development.

Secondary symbionts affect aphid fitness and the titer of primary symbiont.

Bacterial symbionts associated with aphids are important for their ecological fitness. The corn leaf aphid, Rhopalosiphum maidis (Fitch), is one of the most damaging aphid pests on maize and has been reported to harbor Hamiltonella defensa and Regiella insecticola while the effects of the secondary symbionts (S-symbionts) on host ecology and primary symbiont Buchnera aphidicola remain unclear. Here, four aphid strains were established, two of which were collected from Langfang - Hebei Province, China, with similar symbiont pattern except for the presence of H. defensa. Two other aphid strains were collected from Nanning - Guangxi Province, China, with the same symbiont infection except for the presence of R. insecticola. Phylogenetic analysis and aphid genotyping indicated that the S-symbiont-infected and free aphid strains from the same location had identical genetic backgrounds. Aphid fitness measurement showed that aphid strain infected with H. defensa performed shortened developmental duration for 1st instar and total nymph stages, reduced aphid survival rate, offspring, and longevity. While the developmental duration of H-infected strains was accelerated, and the adult weight was significantly higher compared to the H-free strain. Infection with R. insecticola did not affect the aphid's entire nymph stage duration and survival rate. As the H-strain does, aphids infected with R. insecticola also underwent a drop in offspring, along with marginally lower longevity. Unlike the H-infected strain, the R-infected strain performed delayed developmental duration and lower adult weight. The B. aphidicola titers of the H-infected strains showed a steep drop during the aphid 1st to 3rd instar stages, while the augmentation of B. aphidicola titers was found in the R-infected strain during the aphid 1st to 3rd instar. Our study investigated for the first time the effect of the S-symbionts on the ecology fitness and primary symbiont in R. maidis, indicating that infection with secondary symbionts leads to the modulation of aphid primary symbiont abundance, together inducing significant fitness costs on aphids with further impact on environmental adaptation and trophic interactions.

Molluscan RXR Transcriptional Regulation by Retinoids in a Drosophila CNS Organ Culture System.

Retinoic acid, the active metabolite of Vitamin A, is important for the appropriate development of the nervous system (e.g., neurite outgrowth) as well as for cognition (e.g., memory formation) in the adult brain. We have shown that many of the effects of retinoids are conserved in the CNS of the mollusc, Lymnaea stagnalis. RXRs are predominantly nuclear receptors, but the Lymnaea RXR (LymRXR) exhibits a non-nuclear distribution in the adult CNS, where it is also implicated in non-genomic retinoid functions. As such, we developed a CNS Drosophila organ culture-based system to examine the transcriptional activity and ligand-binding properties of LymRXR, in the context of a live invertebrate nervous system. The novel ligand sensor system was capable of reporting both the expression and transcriptional activity of the sensor. Our results indicate that the LymRXR ligand sensor mediated transcription following activation by both 9-cis RA (the high affinity ligand for vertebrate RXRs) as well as the vertebrate RXR synthetic agonist, SR11237. The LymRXR ligand sensor was also activated by all-trans RA, and to a much lesser extent by the vertebrate RAR synthetic agonist, EC23. This sensor also detected endogenous retinoid-like activity in the CNS of developing Drosophila larvae, primarily during the 3rd instar larval stage. These data indicate that the LymRXR sensor can be utilized not only for characterization of ligand activation for studies related to the Lymnaea CNS, but also for future studies of retinoids and their functions in Drosophila development.

Effect of temperature on life cycle and behaviour of Aedes aegypti (Diptera: Culicidae)

In this study, effect of increasing temperature on life cycle, mortality, and behavior of the mosquito species, Aedes aegypti was evaluated. Heat shock was applied at 32°C, 37°C and 42°C for 20 minutes, 1 hour and 2 hours for each temperature to egg, 2nd instar, 3rd instar and pupal stage separately and changes in life cycle pattern was recorded on a routine basis. The control temperature was 27°C at which the mosquito was reared. When heat-shock was applied to eggs, the developmental period for each stage was found to be inversely proportional to temperature rise. The shortest embryonic developmental period was recorded at 32°C, and the shortest larval and pupal stages were recorded at 37°C (2h). The shortest full development period was also found to be at 37°C (2h). No hatching was recorded at 42°C. While heat shock was applied to 2nd instar larvae, 3rd instar larvae and pupae, similar decreased pattern was observed. In this study, 100% viability was observed upon heat shock to eggs, larvae (2nd and 3rd instar) and pupae at 27°C and 32°C. In case of 37°C, egg, 2nd instar and pupal viability decreased. No egg hatched at 42°C, while few 2nd instars survived. Lowest viability rate for 3rd instar larvae and pupae were counted at 42°C. Change in the rate of movement also decreased gradually with increasing temperatures in pupae. The change was irregular in case of 2nd and 3rd instar larvae. The highest movement was recorded for 2nd and 3rd instar larvae at 32°C and 27°C, respectively and lowest was recorded at 42°C. Bangladesh J. Zool. 49 (3): 369-379, 2021

Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila.

Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. Here, we report the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell-based RNAi screen identified 537 candidate kkv regulators on a genome-wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and we identified six genes as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy-related 101(Atg101) was knocked down in Drosophila at the 3rd-instar larval stage; whereas we observed disoriented taenidial folds on larval tracheae when Lines (lin) or Autophagy-related 3 (Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr, and Abd-A showed that the potential complex mechanism of kkv transcription is regulated by an interaction network with bithorax complex components. Our study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis.

New insights into the life history of Monochamus saltuarius (Cerambycidae: Coleoptera) can enhance surveillance strategies for pine wilt disease

Pine wilt disease was first discovered in Dongtang town, Liaoning Province, China, in 2017. Monochamus saltuarius Gebler is a new vector of pinewood nematode and the only known vector in Liaoning Province. The biology of this pest has not been reported thus far; therefore, it is necessary to study its life history. During 2018 and 2019, we collected 138 and 491 adult M. saltuarius beetles, respectively, to analyze their eclosion from larva to adult stage. In mid-March, overwintering larvae began to feed (on xylem) and seek nutrition in preparation for pupation and eclosion. The adults began to appear in mid-April, and the population reached its peak in late May. The life span of the adults was 28–76 days. After approximately 1 week of supplemental nutrition (feeding on twigs), adults began to mate and lay eggs. The egg stage of M. saltuarius lasted 4–8 days. The larvae in Dongtang town have 4 instars and overwinter in tunnels as 3rd–4th instars. The 1st-instar stage lasted 3–9 days, the 2nd-instar stage lasted 11–23 days, the 3rd-instar stage lasted 30–130 days, and the 4th-instar stage lasted 44–180 days. The pupal stage lasted 7–12 days, and the life span of the adults was 28–76 days. In this study we systematically monitored the life history of M. saltuarius for the first time. Our objective was to lay a foundation for improving control of this pinewood nematode vector.

In Vitro Studies for the Evaluation of Insecticidal Potential of the Venom of Endoparasitic Wasp Aenasius arizonensis (Girault) (Hymenoptera, Encyrtidae)

Aenasius arizonensis (Hymenoptera: Encyrtidae) a nymphal, solitary endoparasitoid is a potent biocontrol agent of cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae). Female wasp injects a blend of secretions in its host during parasitization which alters the host physiology. Insecticidal potential of crude venom of A. arizonensis was evaluated along with its chromatographic fractions by microinjections bioassays in its natural host insect P. solenopsis at 3rd nymphal instars and adult. Host insects were treated with two different volumes of crude venom (0.3 and 0.5 µL) and RP-HPLC fractions of venom, while host insects with saline injections were used as control. Biological activity of the crude venom was also evaluated after heat and protease treatment. Maximum mortality (84.0 ± 2.91) of the host insects was observed at highest dose rate (0.5 µL) of the crude venom; while lowest mortality (71.0 ± 2.31) was recorded at lowest dose (0.3 µL) of venom applied on 3rd instar life stage. On the other hand, significant difference in mortality of four peaks of RP-HPLC venom fractions were recorded against 3rd instar larvae and adult when microinjections containing crude venom and fractionated venom. Our data confirms that crude venom of the wasp induces significant mortality against its host P. solenopsis. Whereas, no significant difference in mortality was observed for heat and protease treated venom, suggesting that bioactive component of the venom secretions of the wasp are proteins in nature, which lost their biological activity upon heat and protease treatments.

Makorin 1 Regulates Developmental Timing in Drosophila.

The central mechanisms coordinating growth and sexual maturation are well conserved across invertebrates and vertebrates. Although mutations in the gene encoding makorin RING finger protein 3 (mkrn3 ) are associated with central precocious puberty in humans, a causal relationship has not been elucidated. Here, we examined the role of mkrn1, a Drosophila ortholog of mammalian makorin genes, in the regulation of developmental timing. Loss of MKRN1 in mkrn1exS prolonged the 3rd instar stage and delayed the onset of pupariation, resulting in bigger size pupae. MKRN1 was expressed in the prothoracic gland, where the steroid hormone ecdysone is produced. Furthermore, mkrn1exS larvae exhibited reduced mRNA levels of phantom, which encodes ecdysone-synthesizing enzyme and E74, which is a downstream target of ecdysone. Collectively, these results indicate that MKRN1 fine-tunes developmental timing and sexual maturation by affecting ecdysone synthesis in Drosophila. Moreover, our study supports the notion that malfunction of makorin gene family member, mkrn3 dysregulates the timing of puberty in mammals.

Cloning and characterization of Methoprene-tolerant (Met) and Krüppel homolog 1 (Kr-h1) genes in the wheat blossom midge, Sitodiplosis mosellana.

Juvenile hormone (JH), a growth regulator, inhibits ecdysteroid-induced metamorphosis and controls insect development and diapause. Methoprene-tolerant (Met) and Krüppel homolog 1 (Kr-h1) are two proteins involved in JH action. To gain some insight into their function in development of Sitodiplosis mosellana, an insect pest undergoing obligatory larval diapause at the mature 3rd instar stage, we cloned full-length complementary DNAs of Met and Kr-h1 from this species. SmMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family and eight conserved amino acid residues important for JH binding. SmKr-h1 encoded a protein showing high sequence homology to its counterparts in other species, and contained all eight highly conserved Zn-finger motifs for DNA-binding. Expression patterns of SmMet and SmKr-h1 were developmentally regulated and JH III responsive as well. Their mRNA abundance increased as larvae entered early 3rd instar, pre-diapause and maintenance stages, and peaked during post-diapause quiescence, a pattern correlated with JH titers in this species. Different from reduced expression of SmMet, SmKr-h1 mRNA increased at mid-to-late period of post-diapause development. Topical application of JH III on diapausing larvae also induced the two genes in a dose-dependent manner. Expression of SmMet and SmKr-h1 clearly declined in the pre-pupal phase, and was significantly higher in female adults than male adults. These results suggest that JH-responsive SmMet and SmKr-h1 might play key roles in diapause induction and maintenance as well as in post-diapause quiescence and adult reproduction, whereas metamorphosis from larvae to pupae might be correlated with their reduced expression.

Seasonal and biological interactions between the parasitoid, Aenasius arizonensis (Girault) and its host, Phenacoccus solenopsis Tinsley on cotton

Aenasius arizonensis (Girault) (Hymenoptera: Encyrtidae) is an important solitary endoparasitoid of mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae). Studies on seasonal and biological interactions between host and parasitoid are important to optimize the mass production and field release of high-quality females. We investigated the influence of different crop seasons viz., pre-monsoon, monsoon and post-monsoon and host stages viz., three nymphal instars (1st, 2nd and 3rd) and adult stage of P. solenopsis on the development, longevity, fecundity and sex ratio of its parasitoid, A. arizonensis. A. arizonensis female parasitized all life stages of its host except the 1st instar nymphs. Only males emerged from parasitized 2nd instar nymphs, while the sex ratio in the later host instars was strongly female-biased. The adult host stage was most suitable for A. arizonensis in terms of shorter development time, while the 3rd instar nymphal stage was most suitable with respect to higher fecundity, proportion of adults emerged and more females in the progeny. Among different seasons, post-monsoon season (September–October) with temperature (25 to 29 °C) and relative humidity (68 to 73%) was most suitable for the overall fitness of the parasitoid. The findings of this study have implications in designing mass rearing of this parasitoid and devising an effective biological control strategy for P. solenopsis on cotton.

Evaluation of Four Entomopathogenic Nematodes as Biological Control Agents Against The Housefly, Musca domestica L. (Diptera: Muscidae)

The house fly, Musca domestica L. is considered as a notorious pest by medical, veterinary, and public health professionals worldwide. This study was carried out to evaluate the effect of different entomopathogenic nematode (EPNs) strains against the 3rd larval instar and pupal stage of the house fly. Two of them were native strains (Heterorhabditis indica and Steinernema carpocapsae) and the others were imported strains Heterorhabditis bacteriophora and Steinernema glaseri. The results indicated that the mortality of all tested stages of M. domestica were significantly influenced by the infective juveniles (IJs) concentrations and the exposure time where the maximum mortality was recorded 72 hrs. post-treatment and the larvae were more susceptible to nematodes pathogenicity than the pupae. Also, the Heterorhabditis nematodes H. baceriophora and H. indica appeared to be more effective than the Steinernema species, and H. bacteriophora (Hb88) was the most promising strain in management of both larvae and pupae causing the lower LC50 and LC95 (320.4 and 1987IJs/ml) for larvae and (1414.6 and 2664.4 IJs/ml) for pupae, respectively.

RNA Interference Mediated Serine Protease Gene (Spbtry1) Knockdown Affects Growth and Mortality in the Soybean Pod Borer (Lepidoptera: Olethreutidae)

Abstract The soybean pod borer, Leguminivora glycinivorella Matsumura (Lepidoptera: Tortricidae), is an economically significant soybean pest in northeastern Asia. Serine proteases are crucial enzymes responsible for protein digestion in herbivorous lepidopterans. In this study, a gene (Spbtry1) encoding a soybean pod borer serine protease was cloned from the organism's midgut. The Spbtry1 open reading frame encoded a 269 amino acid protein with a predicted molecular mass of approximately 29 kDa. Alignment of Spbtry1 with trypsins and chymotrypsins from other insects revealed a high degree of conservation in the putative catalytic domain region. Analysis by reverse transcriptase polymerase chain reaction indicated that Spbtry1 was specifically expressed in the midgut, its transcript existed constitutively in the larval stage, and its expression was highest in the 3rd instar larval stage. RNA interference indicated that Spbtry1 expression levels decreased on diets containing Spbtry1 double-stranded RNA (ds...

COMPARISON OF ANTIMICROBIAL, LARVICIDAL AND ANTHELMINTIC ACTIVITY OF CURCUMA AROMATICA SALISB. COW URINE EXTRACT

The present study was undertaken to evaluate and compare antimicrobial, larvicidal and anthelmintic activity of Curcuma aromatica Salisb. cow urine extracts. The antimicrobial activity was performed using five microbial strains, v.i.z, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Aspergillus niger and Aspergillus flavus; larvicidal activity was evaluated using 3rd and 4th instar stage larvaes, and anthelmintic activity using red earthworms (Lumbricus rubellus) with raw and photoactivated cow urine. Antimicrobial activity of photoactivated cow urine extract of C. aromatica (CAPUE) showed significant results when compared with raw cow urine extract of C. aromatic (CAUE). Larvicidal activity of CAPUE at a concentration of 25% V/V i.e. 40.2% V/V was found to show better mortality rate of larvae when compared with CAUE at a concentration of 25% v/v i.e. 45.2% V/V. Anthelmintic activity of CAPUE was found to show better paralysis and death time at a concentration of 10 μg/mL i.e 36.3 min for paralysis and 58.9 min for death when compared to CAUE i.e. 38.3 min paralysis rate at a concentration of 10 μg/mL and death rate is found 69.9 min at 10 μg/mL concentration.

Parasitic Effects of Solitary Endoparasitoid, <i>Aenasius bambawalei </i>Hayat (Hymenoptera: Encyrtidae) on Cotton Mealybug, <i>Phenacoccus solenopsis </i>Tinsley (Hemiptera: Pseudococcidae)

Parasitization has an enormous impact on host physiology, development and reproduction. The effect of parasitism by endoparasitoid, Aenasius bambawalei Hayat (Hymenoptera: Encyrtidae) on survival and reproduction of mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) on different host stages was studied. Our data reveals that host mealybugs parasitized by the wasp at the 2nd instar stage were died during the 3rd instar stage of their life. However, those parasitized at the 3rd instar stage could reach the adult stage and were able to produce their progeny. After 6 days of parasitization, all parasitized hosts of the 2nd, 3rd and adult stages were died except the 1st instar. Results showed that parasitized host mealybugs had significantly lower reproductive potential than the unparasitized ones. Maximum parasitoid emergence was recorded in the 3rd instar host stage. This basic research regarding survival and reproduction of the parasitized host mealybugs would be very helpful in devising sustainable biological control strategies for cotton mealybug.

Assessing dengue outbreak areas using vector surveillance in north east district, Penang Island, Malaysia

Objective To understand the efficacy of ovitrap surveillance and its implementation on monitoring reflection upon case occurrence in relation to climate variables. Methods We used routinely setup ovitrap surveillance to monitor the mosquito populations in previous outbreak areas. Ovitraps were installed weekly at three localities that experienced high number of dengue cases (Flat Hamna, Kampung Sungai Gelugor and Kampung Tanjung Tokong) from January 2010 to February 2011. Ovitraps and paddles were brought back to the laboratory and all of the water contents were poured into an enamel pan. Aged tap water was added into the enamel pan and eggs were allowed to hatch. The hatching larvae were counted after 3 days. The hatched larvae were identified at the 3rd instar larval stage. The ovitrap indices and mean number of larvae were analyzed using student t-test and One-way ANOVA. Spearmen's rank correlation coefficient was used to determine the relation between meteorology variables and dengue fever cases. Results Aedes albopictus was found as dominant species followed by Aedes aegypti recorded in all three study areas. Aedes aegypti preferred to breed outdoor with larvae collection, which was higher than indoor (72.37%). There was a positive correlation between the ovitrap index with the rainfall and humidity except in Kampung Tanjung Tokong. Our result also showed negative correlation between temperature and ovitrap index in all localities. Conclusions This study provides useful data to be adapted in dengue vector management. It is very important to understand the fluctuation of vector population according to the seasonal activity, which can help us to improve our control programs. However, other factors might also contribute to the increment of dengue outbreak such as the number of available breeding sites, behavior of the vector against environmental factors and the cleanliness of the environment.

Altered differential hemocyte count in 3rd instar larvae of Drosophila melanogaster as a response to chronic exposure of Acephate.

Acephate, an organophosphate (OP) pesticide, was used to investigate the effects of its chronic exposure on hemocyte abundance in a non-target dipteran insect Drosophila melanogaster. For this purpose, six graded concentrations ranging from 1 to 6 μg/ml were selected, which are below the reported residual values (up to 14 μg/ml) of the chemical. 1st instar larvae were fed with these concentrations up to the 3rd instar stage and accordingly hemolymph smears from these larvae were prepared for differential hemocyte count. Three types of cells are found in Drosophila hemolymph, namely, plasmatocytes, lamellocytes and crystal cells. Plasmatocyte count was found to decrease with successive increase in treatment concentrations. Crystal cells showed an increasing trend in their number. Though the number of lamellocytes was very low, a bimodal response was noticed. Lamellocyte number was found to increase with the initial three concentrations, followed by a dose dependent reduction in their number. As hemocytes are directly linked to the immune system of fruit flies, fluctuations in normal titer of these cells may affect insect immunity. Hemocytes share homologies in their origin and mode of action with the immune cells of higher organisms including man. Thus the present findings suggest that immune cells of humans and other organisms may be affected adversely under chronic exposure to Acephate.