Application of gene expression of insulin signaling pathway to insect growth modeling

Md. Abdullah Al Baki,Yonggyun Kim 한국응용곤충학회 2017 한국응용곤충학회 학술대회논문집 Vol.2017 No.10

Insect growth depends on temperature and nutrient. Intake nutrients activate insulin signaling pathway, which mediatesthe nutrient signal to coordinate growth in entire body. A subtropical species, Maruca vitrata (Lepidoptera: Crambidae),gives serious damages on various Fabaceae crops. This study predicted seven components (InR, IRS, PI3K, PTEN, Akt,mTOR, FOXO) of the insulin signal and showed that some of the insulin gene expression levels are highly correlatedwith developmental rates. These correlations may be applied to amend a temperature-dependent growth modeling of M.vitrata.

Validation of insulin signaling pathway using CRISPR-Cas9 in Maruca vitrata

Md. Abdullah Al Baki,Yonggyun Kim 한국응용곤충학회 2019 한국응용곤충학회 학술대회논문집 Vol.2019 No.04

Insulin/IGF signaling (IIS) regulates multiple physiological processes such as larval growth, reproduction, and life span in many organisms including legume pod borer, Maruca vitrata (Lepidoptera: Crambidae). RNA interference of IIS components, insulin receptor (InR) and Forkhead Box O (FOXO), impaired larval growth and female reproduction. To further validate the physiological roles of InR and FOXO, we generated knock-out (KO) mutants using CRISPR/Cas9-mediated genome-editing technology. Both KO mutants exhibited delayed larval growth and reduced pupal and adult body sizes. In conclusion, these results demonstrated the critical role of insulin signaling (IIS) pathway to control M. vitrata growth and development.

Regulation of hemolymph trehalose titers by insulin signaling in the legume pod borer, <i>Maruca vitrata</i> (Lepidoptera: Crambidae)

Al Baki, Md. Abdullah,Jung, Jin Kyo,Kim, Yonggyun Elsevier 2018 Peptides Vol.106 No.-

<P><B>Abstract</B></P> <P>A disaccharide, trehalose, is a main hemolymph sugar of the legume pod borer, <I>Maruca vitrata</I> larvae, but its titers fluctuated with feeding activity. During diurnal feeding in the photophase, hemolymph trehalose remained at a relatively low level (69 mM) and increased (98 mM) during scotophase. Starvation significantly increased the hemolymph trehalose level, in which the elevation of trehalose titers was dependent on the non-feeding period. The down-regulation of the trehalose level during the active feeding period seemed to result from mediation of the insulin/IGF signal (IIS). Injection of a porcine insulin suppressed the trehalose level in a dose-dependent manner. Genes associated with IIS of <I>M. vitrata</I> were predicted from its larval transcriptome, and their expression was confirmed in different developmental stages and tissues. All seven IIS genes selected were expressed in all developmental stages and different tissues. Silencing of four IIS genes (insulin receptor, Forkhead box O, a serine-threonine protein kinase, target of rapamycin) by RNA interference significantly modulated the hemolymph trehalose level. Starvation treatment changed expression of two trehalose metabolism-associated genes (trehalose phosphate synthase (TPS) and trehalase (TRE)) as well as the IIS genes. Silencing of <I>TPS</I> or <I>TRE</I> expression significantly down- or up-regulated the hemolymph trehalose level, respectively. In addition, silencing of IIS genes altered both <I>TPS</I> and <I>TRE</I> expression, indicating a functional link between IIS and trehalose metabolism. These results suggest that nutrients obtained from feeding activate IIS of <I>M. vitrata</I>, which then down-regulates the hemolymph trehalose level by altering trehalose metabolism.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An ILP/IGF signal (IIS) pathway is predicted from <I>Maruca vitrata</I>. </LI> <LI> Feeding rhythm coincides with IIS expression to maintain plasma trehalose level. </LI> <LI> RNA interference of IIS gene expression resulted in significant increase of plasma trehaloe level. </LI> </UL> </P>

Insulin/IGF signaling regulates female reproduction by stimulating stem cells in germarium

Md. Abdullah Al Baki,Yonggyun Kim 한국응용곤충학회 2018 한국응용곤충학회 학술대회논문집 Vol.2018 No.04

Insulin/IGF signaling (IIS) regulates different physiological processes such as metabolism, trehalose level, growth, and reproduction. Four IIS components are identified from the bean pod borer, Maruca vitrata (Lepidoptera: Crambidae). RNA interference (RNAi) of insulin receptor (InR), Forkhead Box O (FOXO), Target of Rapamycin (TOR) or Akt led to ovary dysfunction. Especially, the RNAi treatment significantly reduced the stem cell division in the germarium. However, an addition of a porcine insulin stimulated the cell division. Immature diets significantly influenced on the ovarian stem cell development.

Oocyte and sperm development of the striped fruit fly, Zeugodacus scutellata

Md. Abdullah Al Baki,Yonggyun Kim 한국응용곤충학회 2019 한국응용곤충학회 학술대회논문집 Vol.2019 No.10

Discrimination of different generations of Zeugodacus scutellata using age grading technique and their local genetic variation

김용균,Md. Abdullah Al Baki,권기면,김동순,박계청,안정준 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.3

The striped fruit fly, Zeugodacus scutellata, exhibits generation overlap due to its relatively long lifespan. This study clarified the fly generations by age-grading technique and traced migration of local populations using genetic markers. Male adults were monitored using Cue-lure in two different places (Andong and Guri) of mainland Korea during the entire host cultivating period. They showed two occurrence peaks (from late July to early August and from mid-August to mid-October). A developmental model of Z. scutellata using rate summation method predicted four generations during this period. Pterin amounts in fly heads increased with adult age and indicated at least 3 cycles of age variation during the occurrence period with an additional adult generation after the third cycle. The additional peak was supported by field monitoring using protein diet supplemented with terpinyl acetate, which showed an additional occurrence peak during October. These seasonal populations were monitored through their migration by gene flow analysis using randomly amplified polymorphic DNA (RAPD) markers. In each locality, three seasonal populations at July, August, and September were discrete. Although local populations were genetically discrete at July, they were not separate at late seasons except for Jeju populations. These results indicate the usefulness of the head pterin quantification in age-grading of adult populations and suggest that Z. scutellata occurs at least four generations a year. In addition, gene flow analysis using RAPD suggests frequent movements among local populations along with season progression.

Calcium/calmodulin-dependent protein kinase II of the oriental fruit fly, Bactrocera dorsalis, and its association with rapid cold hardiness

안정준,최경선,Shaw-Yhi Huang,Md. Abdullah Al Baki,아메드 샤비르,김용균 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.4

The oriental fruit fly, Bactrocera dorsalis, is a serious insect pest with diverse host range. Furthermore, its invasive and polyphagous behaviors allow this species to expand its habitats. Recent climate change and increase of international trade/transportation facilitate the species expansion from subtropical to temperate regions. Low temperature during winter appears to be the major factor limiting its expansion to temperate zones in the northern hemisphere. This study reports its remarkable ability in rapid cold-hardening (RCH) along with deep supercooling capacity. A brief exposure to 9 °C significantly enhanced cold tolerance of its larvae, pupae, and adults. RCH took 1–2 h for pupae and adults, although it took 24 h for larvae. Supercooling capacity of pupae was also enhanced by RCH treatment from −13.4 °C to −16.6 °C. To trace genetic factors associated with RCH, calcium/calmodulin-dependent protein kinase II (Bd-CaMKII) was identified from B. dorsalis and their expression in response to RCH treatment was analyzed. Bd-CaMKII possesses three conserved domains of kinase, calmodulin, and oligomerization. Bd-CaMKII is highly homologous to CaMKII of D. melanogaster and other tephritid flies. Expression levels of Bd-CaMKII in the larvae treated with RCH were significantly increased by approximately 5.5 folds compared to those in control larvae. In addition, expression levels of HSP70 and HSP90 were also increased in response to RCH treatment. These results along with previous studies suggest that cold-hardening of B. dorsalis is functionally associated with its supercooling capacity with increased production of cryoprotectants and HSP through regulatory activity of Bd-CaMKII.

전자빔 조사를 이용한 호박꽃과실파리의 불임 수컷 제조 기술과 유전방제 응용

김용균,압둘라알바키,권기면,Kim, Yonggyun,Al Baki, Md. Abdullah,Kwon, Gimyeon 한국응용곤충학회 2020 한국응용곤충학회지 Vol.59 No.1

호박꽃과실파리(Zeugodacus scutellata)는 국내 자생종으로 국제적으로는 검역 대상 과실파리 중 하나로 분류되고 있다. 불임충방사기술(sterile insect release technique (SIT))은 검역 대상 과실파리를 박멸하는 데 이용되어 왔다. 본 연구는 호박꽃과실파리를 대상으로 불임충 제조 기술을 개발하여 SIT 기술로 이 해충에 대한 방제 가능성을 분석하였다. 이에 앞서 먼저 형광현미경 기술로 호박꽃과실파리의 생식관련 기관을 관찰하였다. 다영양실형 난소소관을 갖는 1 쌍의 난소는 약 100 개의 난포를 발달시키며, 각 난포는 난모세포와 영양세포를 지니며 난포세포가 둘러싸는 구조를 지닌다. 난소 발육은 우화 후 10 일이 지나 발육을 시작하고 우화 후 20 일이 되면 난각을 가지고 있는 난모세포를 발달시켰다. 수컷의 경우 성숙된 정소가 우화 직후에 관찰되었으며 수정관에는 운동성이 있는 정자로 채워져 있었다. 불임 수컷을 제조하기 위해 다양한 선량(0~1,000 Gy)의 전자빔을 3~5 일 경과된 번데기에 조사하였다. 200 Gy 세기 전자빔으로 번데기에 조사하면 무처리와 차이 없이 성충으로 발육하였고 이후 정상 암컷과 교미행동을 보였다. 비록 이들 처리 수컷의 교미는 무처리 수컷과 비교하여 큰 차이 없는 산란력을 보였지만 산란된 알들은 부화하지 못했다. 다음으로 200 Gy 조사로 형성된 불임 수컷을 정상 암컷과 수컷이 함께 있는 장소에 방사하였다. 이때 불임충 수컷 은 정상 수컷에 비해 9 배 많은 수로 방사하였다. 이러한 불임충 처리는 차세대 부화율을 현격하게 감소시켰다. 이상의 결과는 200 Gy 세기의 전자빔으로 호박꽃과실파리의 불임충을 제조할 수 있고, 이렇게 형성된 불임충은 SIT 방제에 적용할 수 있다는 것을 제시하고 있다. The striped fruit fly, Zeugodacus scutellata, is endemic in Korea, but it has been regarded as one of the serious quarantine pests throughout the world. Sterile insect release technique (SIT) has been used to eradicate quarantine fruit flies. This study developed a technique to generate sterile males and applied SIT to control Z. scutellata. First of all, the reproductive systems of Z. scutellata adults were examined with fluorescent microscope. Polytrophic ovaries comprises of around 100 follicles with developing oocytes. Each follicle contains an oocyte with several nurse cells and are surrounded with follicular epithelium. Oocyte development began at 10 days after adult emergence (DAE) and formed chorionated oocytes after 20 DAE. On the other hand, male testes were well developed just after adult emergence. The vas deferens was filled with motile sperms. To generate sterile males, different doses (0~1,000 Gy) doses of electron beam were irradiated to 3~5 days old pupae of Z. scutellata. When male pupae were irradiated with electron beam at 200 Gy, they developed and mated with females without any significant difference compared to untreated males. Although the untreated females mated with the 200 Gy-irradiated males laid eggs, no eggs did not hatch. The 200 Gy-irradiated males were then applied to untreated male and female flies in a density ratio of 1:9 (untreated males : treated males). The laid eggs suffered significant infertility. These results suggest that electron beam-irradiated pupae at 200 Gy resulted in male sterility and the resulting males would be applied to SIT.

Monitoring the Oriental Fruit Fly (Bactrocera dorsalis), the Melon Fly (B. cucurbitae), and B. tau Fruit Fly Using Wax Formation Lures

김용균,몰라이맘,압둘라 알바키,안정준,Kim, Yonggyun,Imam, Mollah,Al Baki, Md. Abdullah,Ahn, Jeong Joon Korean Society of Applied Entomology 2018 한국응용곤충학회지 Vol.57 No.1

과실파리류는 국내 60종의 검역 해충 가운데 42종을 차지하고 있다. 이들 대부분은 자연계 산물인 methyl eugenol (ME) 또는 raspberry ketone에 특이적으로 유인된다. 이들 유인물질을 파라핀 왁스를 이용하여 제형화하였고, 이를 대만에서 야외 유인 효과를 검증하였다. 이들 왁스제형은 모두 델타트랩에 설치하고 8월 말경에 모니터링 한 결과 ME 왁스 제형은 오리엔탈과실파리(Bactrocera dorsalis)를 매일 60-80 마리 포획하였다. 반면에 Cuelure 왁스제형은 오이과실파리(Bactrocera cucurbitae)와 B. tau를 유인하였다. 이러한 결과는 본 왁스제형이 이들 검역 해충에 대한 국내 모니터링에 응용될 수 있다는 것을 제시하고 있다. Out of 60 quarantine insect pests in Korea, 42 species are classified into tephritid fruit flies. Most of these fruit flies are attracted to two natural products, methyl eugenol (ME) or raspberry ketone. Paraffin wax has been devised to formulate these lures. The formulated lures were applied to field test in Taiwan to attract quarantine fruit flies. Wax-ME formulation was installed in Delta trap and could attract 60-80 males of B. dorsalis per day during late August, while a wax formulation of Cue-lure (a methylated compound of raspberry ketone) attracted both B. cucurbitae and B. tau. These wax formulations can be applied to monitor these three quarantine species in Korea.

오리엔탈과실파리 유전변이 - 대만 지역 집단변이

김용균,김효일,마히이맘몰라,압둘라알바키,Kim, Yonggyun,Kim, Hyoil,Mollah, Md. Mahi Imam,Al Baki, Md. Abdullah 한국응용곤충학회 2019 한국응용곤충학회지 Vol.58 No.2

본 연구는 국내 금지급 과실파리인 오리엔탈과실파리(Bactrocera dorsalis)에 대한 유전적 변이를 분석하였다. 이를 위해 오리엔탈과실파리가 자생하는 대만 지역을 대상으로 2019년 동일한 시기(3일간: 7월 30일~8월 1일)에 서로 다른 세 지역(타이페이, 타이중, 카오슝)에서 과실파리류를 채집하여 나이 변이 및 미토콘드리아 서열 변이를 각각 비교하였다. 세 지역에서 채집된 오리엔탈과실파리는 1,085마리로서 메틸유제놀 유인제에 모두 유인되었으며, 큐루어 유인제에는 30마리의 오이과실파리(Zeugodacus cucurbitae) 및 1마리의 타우과실파리(Bactrocera tau)만 채집되었다. 단백질먹이 유인제에는 총 6마리가 포획되었으며 이 가운데 오리엔탈과실파리는 1마리가 포함되었으며 나머지는 오이과실파리였다. 오리엔탈과실파리 수컷의 머리에는 테린이 포함되었으며 나이가 증가함에 따라 각 머리에는 $32{\mu}g$에서 $59{\mu}g$까지 테린 함량이 증가하였다. 대만 세 지역의 수컷 집단들은 테린 양에 차이를 나타냈으며, 카오슝 집단이 타이페이와 타이중 집단에 비해 적은 테린 양을 보유하였다. 이들 세 지역 사이에 유전적 거리가 RAPD (random amplified polymorphic DNA)를 이용하여 분석되었으며 타이페이 집단이 타이중 및 카오슝 집단들과 차이를 나타내는 것으로 나타났다. 유전적 변이는 미토콘드리아의 cytochrome oxidase I (CO-I)과 NADH dehydrogenase I (ND-I)을 각각 비교하였다. CO-I 영역 가운데 360개 염기서열을 비교한 결과 7.8%의 염기서열 변이를 나타냈다. ND-I 영역을 비교한 결과 213개 염기서열 가운데 6.6%의 염기서열 변이를 보였다. 이들 변이 서열을 대만 지역에 발생하는 오리엔탈과실파리의 특이적 SNP (single nucleotide polymorphism) 마커로 개발하는 데 추천한다. This study analyzed genetic variation of the Oriental fruit fly (OFF), Bactrocera dorsalis, which is designated to be a quarantine insect pest in Korea. OFF samples endemic to Taiwan were collected at three different locations (Taipei, Taichung, and Kaohsiung) for three days from July 30 to August 1 in 2018 and assessed in their age and mitochondrial DNA sequence variations. In these places, 1,085 OFF males were collected using methyl eugenol lure while 30 males of Zeugodacus cucurbitae and one male of Bactrocera tau were collected using Cuelure. A protein diet lure attracted 6 flies including one OFF and 5 flies of Z. cucurbitae. Male heads of OFF contained pterin, which increased in contents with age from 32 to $59{\mu}g/head$. There was a local variation in pterin amounts in OFF heads, in which Kaohsiung population had lower amounts of pterin than Taipei and Taichung populations. Genetic distance among these three populations were measured by random amplified polymorphic DNA and showed that Taipei population was separated from Taichung/Kaohsiung cluster. Genetic variation was also analyzed in sequence variations in cytochrome oxidase I (CO-I) and NADH dehydrogenase I (ND-I). There was 7.8% variation in CO-I sequence (360 residues) and 6.6% variation in ND-I sequence (213 residues). These polymorphic sites are proposed to be used to develop SNP (single nucleotide polymorphism) markers characteristic to Taiwan OFF populations.