Honey bees are challenged with declining colony numbers, generally called ‘Colony Collapse Syndrome’ (CCS). This issue is certainly a syndrome because it is due to a range of threats, including parasitic mites, exposure to agricultural chemicals and various viruses, among other things. We posed the hypothesis the CCS could also be due to declining nutritional qualities, which may relate to digestive physiology. Because there is no information on honey bee lipid nutrition and digestion, we characterized a digestive phospholipase A2 in honey bee midguts. In this paper, I will report on some of our findings. With a focus on students, I will also use this report to share thoughts on presenting and writing for international audiences.

Phospholipase A2 (PLA2) hydrolyzes fatty acids from phospholipids at sn-2 position. Two immune-associated PLA2s are known in Spodoptera exigua. Both are calcium-independent cellular PLA2 (iPLA2A and iPLA2B). These iPLA2s are inducible to immune challenge using different epitopes such as virus, Gram+, Gram- bacteria, and fungi. However, their expression profiles are different between these two iPLA2s. Especially, immune challenge with Gram+ bacteria and fungi significantly up-regulated these iPLA2 expression, suggesting their expression under Toll signal pathway. RNA interferences of Toll or IMD signal components are being investigated.

The belief that honey bee venom (BV) can be used to treat certain immune-related diseases, such as arthritis and rheumatic conditions, goes back to antiquity. A growing number of reports have demonstrated that BV contains at least 18 pharmacologically active components, including phospholipase A2 (PLA2). Recent research has shown that bee venom PLA2 (bvPLA2) induces protective immune responses against several diseases including asthma, Parkinson’s disease, and drug-induced organ inflammation. However, the antiviral properties of bvPLA2 have not been well investigated. Hence, we examined the potential inhibitory effects of bvPLA2 and its possible mechanism of action against a broad panel of pathogenic viruses in vitro. Pre-treatment with bvPLA2 significantly inhibited the replication of vesicular stomatitis virus (VSV), coxsackie virus (H3), enterovirus-71 (EV-71), herpes simplex virus (HSV) and Adenovirus (AdV) dramatically. However, bvPLA2 did not show antiviral activity against Influenza A virus (PR8) and Newcastle disease virus (NDV). Such inhibitory effects were explained by blocking of the attachment of the virus to cells upon bvPLA2 treatment. Additionally, we observed that Heparan sulfate (HS) has an inhibitory effect on the attachment of HSV to the cell surface dose dependently, which was inconsistent with bvPLA2 treatment. These findings suggest that bvPLA2 has an inhibitory effect on the replication of diverse viruses by blocking their attachment to the cell surface and could be a promising source of natural antiviral agents.

아이코사노이드는 탄소수 20 개의 다가불포화지방산 산화물로 구성된다. 이들 다가불포화지방산의 생합성 전구물질을 탐지하기 위해 파 밤나방(Spodoptera exigua)의 서로 다른 조직으로부터 지방산을 분리하여 GC/MS로 조성을 분석하였다. 파밤나방 5령 유충에서 소화관, 지방체, 혈구 및 체벽을 분리하고, 각 조직에서 지질을 추출하여 각각 중성지질, 당지질 및 인지질로 분리하였다. 대부분의 조직은 palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) 그리고 linolenic acid (18:3)를 주요 지방산으로 함유하였다. 그러나 이들 지방산의 조성은 조직과 지질 종류에 따라 상이하였다. 지방체와 혈구세포는 이들 주요 지방산 이외에 myristic acid (14:0)와 3 종류의 미동정 지방산들이 추가로 검출되었다. 서로 다른 지질 종류 가운데 인지질은 중성지질이나 당지질에 비해 상대적으로 높은 linolenic acid를 지닌 반면 포화지방산의 함유량은 낮았다. 전체 불포화지방산의 조성도 조직과 지질 종류에 따라 상이하였다. 인지질은 지방체, 혈구 및 소화관에서 높은 불포화지방산 함유 량을 나타냈다. 세포성 인지질분해효소인 calcium-independent phospholipase A2 (iPLA2)는 지방산 조성을 조절하는 데 역할을 담당하였다. 이 유전자의 RNA 간섭은 중성지질과 인지질에서 지방산 조성의 변화를 유발하였다. 본 연구는 아이코사노이드 생합성의 전구물질로 여겨지는 아라키도닉산을 검출하지 못했다. 이는 곤충에 있어서 아이코사노이드는 포유동물과는 다른 새로운 생합성 과정을 통해 형성되는 것으로 추정된다.

Phospholipase A2 (PLA2) catalyzes an ester hydrolysis at sn-2 position of phospholipids. Various PLA2 genes are classified into at least 15 groups. However, on the basis of physiological functions, PLA2 genes are classified into calcium dependent cellular PLA2 (cPLA2), calcium independent cellular PLA2 (iPLA2) and secretary PLA2 (sPLA2). In insects, several sPLA2 genes are known to be associated with venom or immune functions. However, no known cellular PLA2 genes are identified. This study reports an iPLA2 (SeiPLA2) encoded in Spodoptera exigua. SeiPLA2 has an open reading frame of 2448 bp encoding a sequence of 816 amino acid residues. Its predicted protein is 89.55 KDa and 6.15 pI. SeiPLA2 is expressed in egg, larva, pupa and adult stages. In larval stage, SeiPLA2 is expressed in hemocytes, fat body, epidermis, gut, malpighian tubules and salivary gland. To understand its physiological function, its RNA interference is under investigation.

곤충병원성세균 Xenorhabdus nematophila (Xn)의 일부 대사물질은 대상 곤충의 phospholipase A2 (PLA2)를 억제하여 아이코사노이드 생합성 활성을 저해시킨다. 그러나 이들 세균 대사물질이 억제하는 곤충의 PLA2에 대해서는 알려져 있지 않다. Xn의 배양액에서 화학구조가 동정된 8 가지 대사물질들은 두 종의 나비목 배추좀나방(Plutella xylostella)과 파밤나방(Spodoptera exigua)의 유충에 대하여 살충 활성을 보유했다.특별히 이들 물질은 모두 Bacillus thuringiensis (비티)의 살충력을 크게 향상시켰다. 파밤나방의 세포성 인지질 분해효소(SecPLA2)를 클로닝하고 대장균에서 과발현시켰다. 분리된 SecPLA2를 지방체에서 얻은 인지질과 반응시켰을 때 여러 다가불포화지방산을 해리시켰다. 이 효소활성이 Xn 유래 대사물질들에 의해 뚜렷이 억제되었다. 또한 SecPLA2에 대한 억제효과와 비티 살충력 상승효과 사이에 정의 상관관계를 보였다. 본 연구는 SecPLA2가 Xn 대사물질의 억제 대상 분자 종말점 가운데 하나라고 제시하고 있다.

Among bee venom proteins, phospholipase A2 (PLA2) is critical one of bee venom components to defend against predators intruders. In this study, PLA2 gene from cDNA libarary using the venom glands of Bombus ignitus worker bees(BiVn-PLA2) was cloned and characterized. BiVn-PLA2 spans 2211 bp and consists of three introns and four exons encoding 180 amino acid residues. BiVn-PLA2 shares high levels of identity with a bumblebee, B. terristris (89% protein sequence identity), B. pennsylvanicus (88%), and a honey bee, Apis mellifera (53%). Northern blot analysis revealed that BiVn-PLA2 is expressed in venom gland, indicating that BiVn-PLA2 is one of the venom components of B. ignitus. To determine BiVn-PLA2 of venom components from venom sac, N-terminal amino acid sequencing of a putative BiVn-PLA2 (the purified 18 kDa) was performed by Edman degradation. The N-terminal amino acid sequencing of the 18 kDa protein was coincident with the N-terminal amino acid residues of the mature BiVn-PLA2 and the 18 kDa protein catalysed the hydrolysis of DBPC subs trate[1-O-(6-Dabcyl-aminohexanoyl)-2-O-(12-(5-B ODIPY-entanoyl) aminododecanoyl)-sn-glyceryl phosphatidylcholine] that is a sensitive fluorogenic probe for PLA2 activation. Western blot analysis revealed that BiVn-PLA2 is expressed in the venom gland, stored in the venom sac, and then emitted throughout sting apparatus. Finally, to test BiVn-PLA2 toxicity, BiVn-PLA2 was adjusted to a insect cell (Sf9) at different concentrations (1-30 μg/2×105 cells). The apoptotic cell death assay results showed that the cell survival decreased with increasing concentrations (1-30 μg/2×105 cells).

A monoterpenoid, benzylideneacetone (BZA), is synthesized by an entomopathogenic bacterium, Xenorhabdus nematophila K1, and known to suppress insect immune responses by inhibiting phospholipase A2(PLA2). This was designed to test its effect of insect digestion by oral administration. The beet armyworm, Spodoptera exigua, was tested by treating its artificial diet with different doses of BZA. The second instar larval were treated with the diets and monitored in their pupation, pupal weight, and adult emergence. BZA gave significant adverse effects on the larval development and subsequent adult metamorphosis. Digestive lumen of the fifth instar larval of S. exigua possessed activity, which was significant inhibited by BZA. These results support that BZA can be developed as a novel feeding deterrent.

Phospholipase A2 (PLA2) is the committed catalytic step of eicosanoid biosynthesis, which has been a common molecular target of several entomopathogens to induce insect immunosuppression. Despite critical importance of PLA2 in insect immunity, its gene structure was not known. This study identified insect PLA2 gene associated with immune reactions in the red flour beetle, Tribolium castaneum. Based on a previous study that an immune-associated PLA2 in insect is secretory type of PLA2 (sPLA2), five highly matched cDNA sequences were obtained from T. castaneum genome database using an sPLA2 sequence probe encoded in Drosophila melanogaster. The expressions of these five putative PLA2 were confirmed by reverse transcriptase-polymerase chain reaction. Out of five genes, one PLA2 gene called TcPLA2B was chosen because it showed specific expression in hemocyte and fat body. TcPLA2B was cloned and expressed in Escherichia coli and its protein was purified. The purified TcPLA2B showed PLA2enzyme activity, which was specifically inhibited by bromophenacyl bromide (a specific sPLA2inhibitor) and dithiothreitol (reducing agent of disulfide bond). It was sensitive to pH (optimum at pH 6.0) and reaction temperature (optimum at 10-30°C), and calcium dependency. An immunofluorescence assay indicated that TcPLA2B was localized near to cellular membrane of the cytosol in the hemocytes of T. castaneum at immune chanlenge. Double-stranded RNA (dsRNA) of TcPLA2B-treated larvae showed knockdown of its mRNA expression and did not form hemocyte nodule formation, while control larvae could exhibit time- and bacterial dose-dependent nodule formation in response to bacterial challenge. Addition of arachidonic acid (the catalytic product of PLA2) to the dsRNA-treated larvae rescued the inhibition of nodule formation. These results suggest that TcPLA2B gene is associated with insect immune reaction.

We checked the presence of phospholipase A2(PLA)2 which could split the ester bond at the position 2 in the glycerol backbone of glycerophospholipids, in the cells of hyperthermophiles of Pyrococcus horikoshii and Sulfolobus acidocaldarius. The results obtained are as follows; (1). Pyrococcus horikoshii cells were grown in obligate anaerobic conditions at 95℃ and they needed sulfur as energy source instead of oxygen, while Sulfolobus acidocaldarius species grew well in the aerobic medium (pH 2.5) containing yeast and sucrose at 75℃. (2). Pyrococcus horikoshii cells produced phospholipase A2 in the cell culture media although this species did not show lipase activity at least in the pH range of 1.5 ~ 3.5. Sulfolobus acidocaldarius cells produced lipase hydrolyzing triacylglycerols such as triolein, but did not split any kind of phospholipids used as substates. (3). The compound of 1-decanoyl-2-(p-nitrophenylglutaryl) phosphatidylcholine was not suitable for a substrate in this experiment, though frequently used as a subtrate for checking presence of phospholipase A2, for its decomposi-tion in this experiment. The L-α-phosphatidylcholine-β-[N-7-nitrobenz-2-oxa-1, 3-diazol]aminohexanoyl-γ-hexadecanoyl labelled with a fluorescent material, did not show any migration of acyl chains in the molecule during the reaction with phospholipase A2 under a hot condition. (4). Phospholipase A2 in the cells of Pyrococcus horikoshii, showed the optimum activity at pH6.7~7.2 and 95~105℃, respectively, and was activated by addition of calcium chloride solution. Andthe phospholipase A2 specifically hydrolyzed glycero-phospholipids such as phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and phosphatidyl inositol, but could not split phospholipid containing ether bonds in the molecule such as DL -α-phosphatidylcholine-β-palmitoyl-γ-O-hexadecyl, DL-α-phosphati- dylcholine-β- oleoyl-γ-O-hexadecyl, DL-phosphatidylcholine-dihexadecyl.